U.S. patent number 10,975,159 [Application Number 16/476,171] was granted by the patent office on 2021-04-13 for compounds binding human cd160 and uses thereof.
This patent grant is currently assigned to ELSALYS BIOTECH. The grantee listed for this patent is ELSALYS BIOTECH. Invention is credited to Alexandre Calcei, Helene Haegel, Thierry Menguy, Caroline Rozan.
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United States Patent |
10,975,159 |
Calcei , et al. |
April 13, 2021 |
Compounds binding human CD160 and uses thereof
Abstract
Novel compounds which bind specifically to human CD160,
including a light chain variable domain (VL), a chosen sequence
defined by SEQ ID No: 14 or SEQ ID No: 13 and a heavy chain
variable domain (VH), a sequence chosen from SEQ ID No: 11, SEQ ID
No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29
or SEQ ID No: 30, fragments thereof or derivatives thereof.
Inventors: |
Calcei; Alexandre (Saint Genis
Laval, FR), Haegel; Helene (Illkirch-Grafenstaden,
FR), Menguy; Thierry (Strasbourg, FR),
Rozan; Caroline (Villeurbanne, FR) |
Applicant: |
Name |
City |
State |
Country |
Type |
ELSALYS BIOTECH |
Lyons |
N/A |
FR |
|
|
Assignee: |
ELSALYS BIOTECH (Lyons,
FR)
|
Family
ID: |
1000005484060 |
Appl.
No.: |
16/476,171 |
Filed: |
January 8, 2018 |
PCT
Filed: |
January 08, 2018 |
PCT No.: |
PCT/EP2018/050354 |
371(c)(1),(2),(4) Date: |
July 05, 2019 |
PCT
Pub. No.: |
WO2018/127586 |
PCT
Pub. Date: |
July 12, 2018 |
Prior Publication Data
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|
|
|
Document
Identifier |
Publication Date |
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US 20200109211 A1 |
Apr 9, 2020 |
|
Foreign Application Priority Data
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|
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Jan 6, 2017 [FR] |
|
|
17/50152 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K
16/464 (20130101); C07K 16/2896 (20130101); C07K
16/468 (20130101); C07K 2317/35 (20130101); C07K
2317/31 (20130101) |
Current International
Class: |
C07K
16/28 (20060101); C07K 16/46 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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2977142 |
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Aug 2016 |
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CA |
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1626059 |
|
Feb 2006 |
|
EP |
|
1776387 |
|
Jul 2010 |
|
EP |
|
98/21240 |
|
May 1998 |
|
WO |
|
2008/155363 |
|
Dec 2008 |
|
WO |
|
2010/084158 |
|
Jul 2010 |
|
WO |
|
2011/147984 |
|
Dec 2011 |
|
WO |
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WO-2014177652 |
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Nov 2014 |
|
WO |
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WO-2017114694 |
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Jul 2017 |
|
WO |
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Other References
Cai et al, "CD160 inhibits activation of human CD4+ T cells through
interaction with herpesvirus entry mediator", 2008, Nature
Immunology vol. 9, pp. 176-185 (Year: 2008). cited by examiner
.
"Antibodies--Structure and Sequence" [online, 2001 archive accessed
from]
[https://web.archive.org/web/20010214233200/http://www.bioinf.org.uk/abs/-
] (Year: 2001). cited by examiner .
CD160 [Homo sapiens]. [online reference] [accessed from:
https://www.ncbi.nlm.nih.gov/protein/CAG46665.1?report=genpept]
(Year: 2008). cited by examiner .
Labrijn et al. "Species-Specific Determinants in the IgG CH3 Domain
Enable Fab-Arm Exchange by Affecting the Noncovalent CH3-CH3
Interaction Strength." J Immunol 2011; 187:3238-3246 (Year: 2011).
cited by examiner .
Ravn et al. "Structural and Pharmacological Characterization of
Novel Potent and Selective Monoclonal Antibody Antagonists of
Glucose-dependent Insulinotropic Polypeptide Receptor". The Journal
of Biological Chemistry vol. 288, No. 27, pp. 19760-19772, Jul. 5,
2013 (Year: 2013). cited by examiner .
Ravn et al. "Chain C, Gipg013 Fab, Antagonizing Antibody to the Gip
Receptor, Heavy Chain" [online] [accessed from
https://www.ncbi.nlm.nih.gov/protein/4HJ0_C] (Year: 2013). cited by
examiner .
Zhang et al. "High levels of CD160 expression up-regulated counts
of chronic lymphocytic leukemia cells and were associated with
other clinical parameters in Chinese patients with chronic
lymphocytic leukemia". Leukemia & Lymphoma, 56:2, 529-532
(Year: 2015). cited by examiner .
"Prevent--Definition and Synonyms" MacMillan Dictionary.
[online][2015 archived version downloaded using the Wayback Machine
from
https://web.archive.Org/web/20150322020300/https://www.macmillandictionar-
y.com/us/dictionary/american/prevent]. (Year: 2015). cited by
examiner .
Safdari et al. "Antibody humanization methods--a review and
update". Biotechnology and Genetic Engineering Reviews, 2013. vol.
29, No. 2, 175-186. (Year: 2013). cited by examiner .
Apr. 17, 2018 International Search Report issued in International
Patent Application No. PCT/EP2018/050354. cited by applicant .
Apr. 17, 2018 Written Opinion issued in International Patent
Application No. PCT/EP2018/050354. cited by applicant .
De Jong et al. "A Novel Platform for the Potentiation of
Therapeutic Antibodies Based on Antigen-Dependent Formation of IgG
Hexamers at the Cell Surface." PLoS Biology: e 1002344, DOI:
10.1371/journal, Jan. 6, 2016, vol. 14, No. 1, pp. 1-24. cited by
applicant .
Diebolder et al. "Complement Is Activated by IgG Hexamers Assembled
at the Cell Surface." Science, Mar. 14, 2014, vol. 343, No. 6176,
pp. 1260-1263, doi: 10.1126/science.1248943. cited by applicant
.
El-Far et al. "CD160 isoforms and regulation of CD4 and CD8 T-cell
responses." Journal of Translational Medicine, 2014, vol. 12, No.
217. cited by applicant .
Gadkar et al. "Design and Pharmacokinetic Characterization of Novel
Antibody Formats for Ocular Therapeutics." Investigative
Ophthalmology and Visual Science, Aug. 2015, vol. 56, No. 9, pp.
5390-5400. cited by applicant .
Krzystolik et al. "Prevention of Experimental Choroidal
Neovascularization With Intravitreal Anti-Vascular Endothelial
Growth Factor Antibody Fragment." Archives of Ophthalmology, Mar.
2002, vol. 120, 338-346. cited by applicant .
Labrijn et al. "Controlled Fab-arm exchange for the generation of
stable bispecific IgG1." Nature Protocols, 2014, vol. 9, No. 10,
2450-2463. cited by applicant .
Wang et al. "Molecular Basis of Assembly and Activation of
Complement Component C1 in Complex with Immunoglobulin G1 and
Antigen." Molecular Cell, Jul. 7, 2016, vol. 63, pp. 135-145. cited
by applicant .
Giustiniani et al. "Identification and characterization of a
transmembrane isoform of CD160 (CD160-TM), a unique activating
receptor selectively expressed upon human NK cell activation."
Journal of Immunology, Jan. 2009, vol. 182, No. 1, pp. 63-71. cited
by applicant.
|
Primary Examiner: Hama; Joanne
Assistant Examiner: Humbarger; Scott T.
Attorney, Agent or Firm: Oliff PLC
Claims
The invention claimed is:
1. A compound which binds specifically to human CD160 and chosen
from the group consisting of antibodies, fragments thereof and
derivatives thereof, the compound comprising: a light chain
variable domain (VL) of SEQ ID No: 14, and a heavy chain variable
domain (VH) chosen from SEQ ID No: 11, SEQ ID No: 25, SEQ ID No:
26, SEQ ID No: 27, SEQ ID No: 28, SEQ ID No: 29 and SEQ ID No:
30.
2. The compound according to claim 1, wherein this compound is a
monoclonal antibody.
3. The compound according to claim 1, wherein it is a monoclonal
antibody which has, as heavy chain constant domain, a sequence
chosen from SEQ ID No: 15, SEQ ID No: 16, SEQ ID Nos 31 to 35, SEQ
ID Nos 43 and 44 and the aglycosylated mutants thereof, and as
light chain constant domain, a sequence chosen from SEQ ID No: 22,
SEQ ID No: 23 and SEQ ID No: 24.
4. The compound according to claim 1, having a light chain defined
by SEQ ID No: 57 and as heavy chain a sequence chosen from SEQ ID
Nos: 45 to 51, SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 12 and SEQ
ID Nos: 58 to 64.
5. The compound according to claim 1, wherein it is a fragment
chosen from a Fab, a Fab' and a F(ab')2, and comprising a light
chain defined by SEQ ID No: 57 and a heavy chain comprising a
sequence chosen from SEQ ID No: ID 36, SEQ ID No: 37 and SEQ ID No:
38.
6. The compound according to claim 1, wherein it is a derivative of
the compound chosen from scFvs, multimeric scFvs fused to an Fc
fragment, diabodies, triabodies, tetrameric scFvs, dimers of which
each monomer comprises an scFv bonded to a heavy chain fragment,
dimers of which each monomer comprises an scFv bonded to heavy
chain fragments, F(ab')2s fused in the C-terminal position to a
leucine zipper domain, single-domain antibodies, forms comprising
at least 2 Fabs bonded head-to-tail and a tetravalent antibody.
7. The compound according to claim 1, wherein it is a
multispecific, or at least bispecific, derivative and that it
comprises at least one CD160-binding site and one site for binding
of another antigen.
8. The compound according to claim 7, wherein the other antigen is
chosen from the following antigens: VEGF-A, VEGF-B, VEGF-C, VEGF-D,
PIGF, VEGF-R2, angiopoietin 2; angiopoietin like 4, CD200R,
PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD, PDGF-R, FGF such as
FGF, beta-amyloid, sphingosine-1-phosphate (S1 P), C'S, IL6, MER
TK, CD1 15, TNF alpha, IL8, HGF, TGF beta, IGF1, IL1, IL2, EGF,
KGF, G-CSF, GM-CSF, alpha-v,beta-3 or alpha-v,beta-5 integrins,
transmembrane or soluble CD146; MMP 1, MMP 2, MMP 9, MT1 -MMP,
TIMP-2; angiogenic PD-ECGF, platelet activation factor;
prostaglandin E, pleiotropin, class II MHC, t HP59, CM101, CD3,
CD25, CD28, PD1, CTLA4, 4-1 BB, LAG-3, ICOS, CD16, CD3, CD47, CD20,
CD19, CD5, CD180, CD200, CD40, CD20, CD37, CD38, CD148, CD180 and
any other antigen specific for B-type lymphomas.
9. A composition comprising at least one compound according to
claim 1.
10. The composition according to claim 9, also comprising at least
one other antibody directed against one of the antigens chosen from
the following antigens: VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF,
VEGF-R2, angiopoietin 2; angiopoietin like 4, CD200, CD200R,
PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD, PDGF-R, FGF,
beta-amyloid, sphingosine-1-phosphate (S1 P), C'5, IL6, MER TK, CD1
15, TNF alpha, IL8, HGF, TGF beta, IGF1, IL1, IL2, EGF, KGF, G-CSF,
GM-CSF, alpha-v,beta-3 or alpha-v,beta-5 integrins, transmembrane
or soluble CD146; MMP 1, MMP 2, MMP 9, MT1 -MMP, TIMP-2;
angiogenin; PD-ECGF, platelet activation factor; prostaglandin E,
pleiotropin, class II MHC, HP59, CM101, CD37, CD38, CD25, CD28,
CD40, PD1, CTLA4, 4-1 BB, LAG-3, ICOS, CD16, CD3, CD47, CD20, CD19,
CD5, CD180, CD200, CD40, CD20, CD37, CD38, CD148, CD180 and any
other antigen specific for B-type lymphomas .
11. A medicament comprising the compound according to claim 1.
12. A product comprising: the compound according to claim 1; and at
least one antibody directed against one of the antigens chosen from
the following antigens: VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF,
VEGF-R2, angiopoietin 2; angiopoietin like 4, CD200, CD200R,
PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD, PDGF-R, FGF,
beta-amyloid, sphingosine-1-phosphate (51 P), C'S, IL6, MER TK, CD1
15, TNF alpha, IL8, HGF, TGF beta, IGF1, IL1, IL2, EGF, KGF, G-CSF,
GM-CSF, alpha-v,beta-3 or alpha-v,beta-5 integrins, transmembrane
or soluble CD146; MMP 1, MMP 2, MMP 9, MT1 -MMP, TIMP-2;
angiogenin; PD-ECGF, platelet activation factor; prostaglandin E,
pleiotropin, class II MHC, HP59, CM101, CD37, CD38, CD25, CD28,
CD40, PD1, CTLA4, 4-1 BB, LAG-3, ICOS, CD16, CD3, CD47, CD20, CD19,
CD5, CD180, CD200, CD40, CD20, CD37, CD38, CD148, CD180 and any
other antigen specific for B-type lymphomas; wherein the product is
for simultaneous, separate or sequential use in the treatment of a
pathological condition which causes a neovascularization chosen
from neovascular ocular pathological conditions, diabetes, diabetic
blindness, primary diabetic retinopathy or age-related macular
degeneration, rheumatoid arthritis, pre-eclampsia, eclampsia or
cancers.
13. The medicament according to claim 11, for use as an
anti-angiogenic, immunomodulator and/or cytotoxic agent.
14. A method for treating a pathological condition, comprising
administering the medicament according to claim 11 to a subject
having the pathological condition, wherein the pathological
condition is chosen from neovascular ocular pathological
conditions, diabetes, diabetic blindness, primary diabetic
retinopathy or age-related macular degeneration, rheumatoid
arthritis, pre-eclampsia, eclampsia or cancers.
15. The method of claim 14, wherein the pathological condition is a
cancer.
16. The method of claim 14, wherein the pathological condition is a
haematological cancer and at least one other anti-CD20 antibody,
anti-CD37 antibody; anti-CD38 antibody or anti-CD40 antibody is
administered to the subject.
17. An isolated nucleic acid encoding the compound according to
claim 1.
18. A vector comprising the nucleic acid according to claim 17.
19. A host cell comprising the vector according to claim 18.
20. A method for producing a compound, comprising culturing the
host cell according to claim 19 so as to produce the compound which
binds specifically to human CD160.
Description
The present invention relates to a compound which binds
specifically to human CD160, having as light chain variable domain
(VL) a sequence defined by SEQ ID No: 14 or SEQ ID No: 13, and as
heavy chain variable domain (VH) a sequence chosen from SEQ ID No:
11, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No: 28, SEQ
ID No: 29 and SEQ ID No: 30, fragments thereof or derivatives
thereof.
Currently, monoclonal antibodies are used as therapies for treating
a variety of pathological conditions, including cancers, autoimmune
diseases, chronic inflammatory diseases, transplant rejection,
infectious diseases, cardiovascular diseases and certain ocular
pathological conditions. There are no fewer than about twenty
monoclonal antibodies or some of the fragments thereof on the
market, and more than four hundred are in clinical development.
The choice of an antibody as a potential candidate in therapy is
therefore of major strategic interest. In particular, the antibody
selected must have a good affinity and good specificity for its
target, optimal efficacy with regard to its possible toxicity,
while at the same time being as non-immunogenic as possible.
Among the existing antibodies which bind specifically to the CD160
receptor is the CL1-R2 antibody. It is the murine monoclonal
antibody directed against the human CD160 receptor described in
patent EP1776387B1. This CL1-R2 antibody has SEQ ID No: 1 as heavy
chain variable domain (VH), and SEQ ID No: 2 as light chain
variable domain (VL). It has anti-angiogenic properties and also
immunomodulatory properties. However, its administration in human
beings is limited, because of its excessive immunogenicity due to a
"HAMA response," referring to the development of human anti-murine
(mouse) antibodies (HAMA), which (i) would induce, in the end,
neutralization (or acceleration of its elimination) of the antibody
and thus of its therapeutic effects and (ii) could also induce a
potential risk of toxicity (adverse immune reactions such as
anaphylaxis or serum diseases).
There is a need to provide compounds, in particular antibodies,
that are effective in the treatment of pathological conditions
involving a neovascularization, in addition to the current
anti-VEGF treatments that are available involving inhibitors of
Vascular Endothelial Growth Factor (VEGF).
Indeed, angiogenesis which is the formation of new blood vessels
from the preexisting vasculature, occurs physiologically. However,
it also plays a role in various pathologies as corneal-retinal
neovascular diseases with mainly ischaemic retinopathies (IR) or
choroidal ones such as exudative or "wet" age-related macular
degeneration (wAMD). Together, they constitute the first cause of
moderate and severe vision loss in developed countries.
Increasing knowledge of angiogenesis and its etiology in wAMD and
IR has led to the development of drugs that target the VEGF
pathway. Intravitreal (IVT) injections of anti-VEGF therapeutic
agents have emerged over the past decade as the first-line
treatments in wAMD, retinal vein occlusion (RVO) and macular edema
(DME).
Even though anti-VEGF therapy appears safe in the general
population, several limitations related to efficacy and safety have
clearly emerged: frequent IVT injections are often required to
reach full efficacy; the prolonged use of anti-VEGF leads to a
reduction of long-term efficacy, linked to tachyphylaxis or
tolerance phenomenon. More than 30% of wAMD patients still poorly
respond and thus are resistant to anti-VEGFs. Furthermore, local
and systemic adverse effects such as increased risk of elevation of
blood pressure, stroke, and myocardial infarctions after repeated
administrations of anti-VEGFs have been reported in patients with
wAMD.
These limitations underline the need to improve the sustain
delivery approaches for anti-VEGFs without increasing the rate of
tolerability or safety issues as well as reducing the number of
injections. The increase of the rate of patient response is
critical to address the large population of poor or no responders.
There is therefore a pressing need to develop VEGF-independent
complementary and synergistic therapies that inhibit pathological
neovascularization while having little or no effect on normal
mature tissue vasculature.
These compounds will have to have good biological activity and
specific affinity for their targets, while at the same time being
well-tolerated and in particular non-immunogenic in human
beings.
There is also a need for new agents which can be combined with the
current treatments, in particular for stimulating effector immune
cells such as NK cells and/or by lifting the anergy of cytotoxic T
cells.
The present invention makes it possible to solve these problems. A
subject of the present invention is a compound which binds
specifically to human CD160 and has as light chain variable domain
(VL) a chosen sequence defined by SEQ ID No: 14 or SEQ ID No: 13
and as heavy chain variable domain (VH) a sequence chosen from SEQ
ID No: 11, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27, SEQ ID No:
28, SEQ ID No: 29 and SEQ ID No: 30, and the fragments or
derivatives of said compound. The compounds of the invention are
specifically suitable for administration in human beings in whom
they are well-tolerated and non-immunogenic.
The compounds of the invention can take the form of an antibody and
more particularly of a monoclonal antibody, of fragments or of
derivatives, and are capable of binding to human CD160, with very
good affinity.
It has been discovered that the compounds according to the
invention exhibit a much better affinity on recombinant CD160
protein and on CD160 positive cells than the parental CL1-R2
antibody or its recombinant chimeric version with same variable
regions and human constant regions. This is very advantageous,
since the chimeric version which could be compatible with an
administration in human beings, still exhibits a certain amount of
potential immunogenicity. In addition, such antibodies and the
fragments and derivatives thereof have excellent activity.
This is evidenced in the following examples, and particular in
Example 1, where the affinity measurements clearly show,
unexpectedly, that the H7, in the IgG1 and IgG4 formats, has a much
better affinity for human CD160 than the murine CL1-R2 and its
respective chimeric human IgG1 and IgG4 forms. The gain in K.sub.D
(see Example 1, Table 1, column 8, K.sub.D gain) compared to the
parental CL1-R2 anti-CD160 K.sub.D is about 3.75 and 3.34 for H7 in
the human IgG1 and IgG4 format respectively. For one and the same
concentration of 50 nM of antibody, a better response for the H7
IgG1 than for H7 IgG4 and CL1-R2 and a worse response for the two
anti-CD160s in the chimeric formats were also obtained (see Example
1, Table 1, column 9).
Furthermore, as described in Example 8, all the compounds according
to the invention have a very different elimination profile in the
bloodstream compared to CL1-R2 and to a representative human IgG1,
the bevacizumab, with a faster clearance in the serum as measured
by systemic pharmacokinetic parameters in rabbit.
The term "human CD160" is intended to mean the human CD160
receptor. It is a 27 kDa receptor which recognizes conventional HLA
molecules (HLA A and C) and non-conventional HLA molecules (HLA G)
and HVEM (Herpes virus entry mediator), anchored to the cell
membrane by a Glycosylphosphatidylinositol (GPI) motif and
belonging to the immunoglobulin superfamily (presence of an
immunoglobulin like domain). This protein is further named CD160
GPI. This protein is physiologically expressed by immune cells: NK
CD56.sup.dim CD16.sup.bright, T CD8 subset, T gamma-delta and T CD4
cells subset. CD160 is also upregulated in pathological conditions
on B cells in hematological cancer as B-CLL or on activated
endothelial cells in neovascular ocular pathologies. The cDNA of
human CD160 corresponds to the sequence SEQ ID No: 1 described in
WO 98/21240. The mRNA of human CD160 is available in Genbank under
accession number AF060981. The protein sequence of human CD160
corresponds to the sequence SEQ ID No: 2 described in WO 98/21240,
and is available under accession number AAC72302 in Genbank.
It should be noted that CD160 protein exists also on another
isoform with a transmembrane (TM) domain hereafter designated as
CD160 TM. The protein sequence of the CD160 GPI isoform protein is
100% homolog with the 76.5% first N terminal part of the CD160 TM
isoform protein sequence. The cDNA of human CD160 TM isoform is
described in WO 2008/155363. The mRNA of human CD160 is available
in Genbank under accession number EU016100.1. The protein sequence
of human CD160 TM is available under accession number ABV89736.1 in
Genbank.
BY55 commercial anti CD160 IgM and CL1-R2 are specific of the CD160
GPI form and are not able to recognize the CD160 TM isoform as
respectively described in (Giustiniani et al, 2009) and (El-Far et
al, 2014). In prior art, there is no anti-CD160 mAb described to
recognize both isoforms of CD160.
It is another advantageous feature of the compounds according to
the invention that they are able to recognize both isoforms of
CD160 (the TM and the GPI) while the parental CL1-R2 antibody is
not and this is to broaden indications when these two isoforms are
present on the same cells (like for example very in the T and NK
lymphomas)
In the context of the present invention, the term "variable region"
or "variable domain" of a compound is intended to mean a region or
domain which relates to the amino-terminal domains of the heavy or
light chain of an antibody. The heavy chain variable domain can be
referred to as "VH". The light chain variable domain can be
referred to as "VL". These domains are generally the most variable
parts of an antibody and contain the antigen-binding sites. This
compound can take the form of an antibody, in particular a
monoclonal antibody.
A light or heavy chain variable region (VL or VH) consists of a
"framework region" interrupted by three hypervariable regions
called "complementarity-determining regions" or "CDRs".
All of the 6 CDRs enable the binding of the antibody to its target
antigen. For example, the CL1-R2 antibody has, as CDRs, the
sequences SEQ ID Nos: 3 to 8 in accordance with the AbM CDR
terminology (broader and adapted to antibody affinity maturation
technologies). These CDRs are present in the compounds H7 of the
present invention.
The compounds according to the invention have excellent affinity
for their target, human CD160, which is greater than that of CL1-R2
or than a chimeric form of this CL1-R2 (cf. Example 1).
Preferably, the compound of the invention has the sequence SEQ ID
No: 11 as heavy chain variable domain (VH), and the sequence SEQ ID
No: 14 as light chain variable domain (VL). Such an antibody
corresponds in particular to the "H7" antibody mentioned in Example
1.
Variants of the H7 antibody have been obtained as described in
example 2.
In another implementation of the invention, the compound has the
sequence SEQ ID No: 25 as heavy chain variable domain (VH), and the
sequence SEQ ID No: 14 as light chain variable domain (VL). Such a
compound corresponds in particular to the "F04" antibody.
In another implementation of the invention, the compound has the
sequence SEQ ID No: 26 as heavy chain variable domain (VH), and the
sequence SEQ ID No: 14 as light chain variable domain (VL). Such a
compound corresponds in particular to the "D09" antibody.
In another implementation of the invention, the compound has the
sequence SEQ ID No: 27 as heavy chain variable domain (VH), and the
sequence SEQ ID No: 14 as light chain variable domain (VL). Such a
compound corresponds in particular to the "A12" antibody.
In another implementation of the invention, the compound has the
sequence SEQ ID No: 28 as heavy chain variable domain (VH), and the
sequence SEQ ID No: 14 as light chain variable domain (VL). Such a
compound corresponds in particular to the "G05" antibody.
In another implementation of the invention, the compound has the
sequence SEQ ID No: 29 as heavy chain variable domain (VH), and the
sequence SEQ ID No: 14 as light chain variable domain (VL). Such a
compound corresponds in particular to the "D12" antibody.
In another implementation of the invention, the compound has the
sequence SEQ ID No: 30 as heavy chain variable domain (VH), and the
sequence SEQ ID No: 14 as light chain variable domain (VL). Such a
compound corresponds in particular to the "A09" antibody.
In one particular implementation of the invention, the compound is
a monoclonal antibody targeting human CD160, which preferably has,
as constant region, a constant region of an immunoglobulin (IgG),
preferably of IgG1 or of IgG4.
The term "constant domain" or "constant region" as defined herein
is intended to mean a constant region derived from an antibody
which is encoded by one of the heavy or light chain immunoglobulin
constant region genes.
The term "constant light chain" or "light chain constant region",
as used in the context of the present invention, is intended to
mean the region of an antibody encoded by the kappa (Ckappa) or
lambda (Clambda) light chains. The constant light chain typically
comprises a unique domain and, as defined herein, refers to
positions 108-214 of Ckappa, or Clambda, where the numbering is
according to the EU index (Kabat et al., 1991).
The term "constant heavy chain" or "heavy chain constant region" is
intended to mean herein the region of an antibody encoded by the
mu, delta, gamma, alpha or epsilon genes in order to define the
isotype as the antibody as IgM, IgD, IgG, IgA or IgE, respectively.
For full-length IgG antibodies, the constant heavy chain, as
defined herein, refers to the N-terminal end of the CH1 domain to
the C-terminal end of the CH3 domain, thus comprising positions
118-447, where the numbering is according to the EU index.
Preferably, the constant region of the compound targeting human
CD160 according to the invention is a constant region of IgG. It
can be chosen from the constant regions of IgG1, IgG2, IgG3 and
IgG4.
Preferably, the constant region of the compound targeting human
CD160 according to the invention is a constant region of IgG1 (SEQ
ID No: 16), or IgG1 E345K (SEQ ID No: 43) or E430G (SEQ ID No: 44)
for indications in oncology, or of IgG4 S228P/R409K (SEQ ID No: 15)
or IgG4-(S228P/R409K)+L235E (SEQ ID No: 31) or IgG1 N297Q or else
the variants IgG4-(S228P/R409K)+H310A/H435Q (SEQ ID No: 32),
IgG4-(S228P/R409K)+I253A (SEQ ID No: 33), IgG1-(N297Q)+H310A/H435Q
(SEQ ID No: 34) and IgG1-(N297Q)+I253A (SEQ ID No: 35) and the
aglycosylated mutants thereof for ophthalmology.
The IgG4 subclass and its variants has very low affinity for the
effectors involved in the complement cascade and Fc gamma receptors
(or FcR including FcgRIIa, FcgRIIIa and FcgRI), which makes it
advantageous in the case where the CDC (complement-dependent
cytotoxicity) effect and/or the ADCC (antibody-dependent
cell-mediated cytotoxicity) effect and/or the ADCP the (antibody
dependent cell phagocytosis) is/are not desired and where it is
desired to limit the possible risks of toxicity at the antibody
obtained.
Conversely, the IgG1 subclass and its variants are responsible for
strong ADCC and/or CDC activity and/or ADCP, which makes them
advantageous for increasing the cytolysis of target cells, but with
a greater risk of toxicity.
In one implementation of the invention, the compound is a
monoclonal antibody targeting human CD160, having as light chain
constant domain a sequence chosen from SEQ ID No: 22 (Km3
polymorphism corresponds to Ala153/Val191), SEQ ID No: 23 (Km1
polymorphism corresponds to Val153/Leu191) and SEQ ID No: 24 (Km1,2
polymorphism corresponds to Ala153/Leu191), and as heavy chain
constant region a sequence chosen from SEQ ID No: 15, SEQ ID No:
16, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34 and
SEQ ID No: 35, and the aglycosylated mutants thereof.
More preferentially, the compound according to the invention
targeting human CD160 has as heavy chain constant domain a sequence
chosen from SEQ ID No: 15, SEQ ID No: 16, SEQ ID No: 31, SEQ ID No:
32, SEQ ID No: 33, SEQ ID No: 34, SEQ ID No: 35, and the
aglycosylated mutants thereof, and as light chain constant domain
the sequence SEQ ID No: 22.
A compound according to the invention can be monospecific or
monofunctional for CD160, while being monovalent (a single
antigen-binding site, in the case in point human CD160), or while
being multivalent (at least 2 sites for binding to human
CD160).
A compound according to the invention can also be a multispecific
compound, for example: a bispecific antibody (bsab) or a similar
molecule. The multispecific compounds are compounds which have
binding specificities for at least two different epitopes, which
are typically non-overlapping. These epitopes can be on identical
or different targets. If the epitopes are on different targets,
these targets can be on the same cell or on different cells or
different cell types. In certain embodiments, one of these binding
specificities is CD160, in particular the extracellular domain of
human CD160, and the other is for another antigen.
A multispecific compound according to the invention can take the
form of bispecific antibodies in the IgG format (i.g. bsab,
orthogonal Fabs, strand exchange engineered domain SEED or
Seed-body), of IgGs with Fabs or ScFvs fragments added (i.g.: DVD
Igs, Dual domain double head antibodies, Di-diabodies, Affibodies,
Biomunex, Fynomab), of bsabs based on antibody fragments (e.g.
bispecific antibody fragments, Fv dimers, BITEs, ImmTACS, DART,
BIKEs), of trispecific antibodies, of bsabs based on fusion
proteins (e.g. scFV-fusions BsAb), of aggregated antibodies,
etc.
The epitopes targeted by the multispecific compounds (i.e. which
are capable of binding specifically to CD160 and to at least one
antigen other than CD160) or targeted by the antibody different
from the compound according to the invention and present in a
composition according to the invention can be present in the
following antigens which are targets of which the activation or
neutralization may have key roles in the inhibition of angiogenesis
or of inflammation associated with this angiogenesis process, such
as the following molecules: VEGF (VEGF-A, VEGF-B, VEGF-C or VEGF-D)
and also PlGF (placental growth factor), VEGF-R2, angiopoietin 2;
angiopoietin like 4, CD200, CD200R, PDGFs (PDGF-AA, PDGF-B,
PDGF-BB, PDGF-CC or PDGF-DD), PDGF-Rs, FGFs such as FGF2 or FGF
beta, beta-amyloid, sphingosine-1-phosphate (S1P), C'5, IL6, MER
TK, CD115, TNF alpha, IL8, HGF, TGF beta, IGF1, IL1, IL2, EGF, KGF,
G-CSF, GM-CSF, alpha-v,beta-3 and alpha-v,beta-5 integrins,
transmembrane and soluble CD146; metalloproteases (such as MMP 1, 2
and 9 and MT1-MMP); TIMP-2; angiogenin; endothelial cell growth
factor (PD-ECGF); platelet activation factor; prostaglandin E;
pleiotropin or the class II MHCs, HP59 or CM101; or targets of
which the activation or neutralization may have key roles in the
re-activation of T lymphocytes, the immunosuppression of which
correlates with a poor prognosis and a progression of cancer, such
as the following molecules: CD3, CD25, CD28, CD40, PD1, CTLA4,
4-1BB, LAG-3 or ICOS, or molecules of which the targeting would
make it possible to get close to the key players of the immune
system of CD160-positive cells, such as the following molecules:
CD16, CD3, CD47, or else molecules of which the targeting would
reinforce the specificity of the bsabs antibody for B lymphomas,
such as the following molecules: CD20, CD19, CD5, CD200 for CLLs,
CD180 for marginal zone lymphomas (MZL) and CD148 for mantle cell
lymphomas, or else antigens which make it possible to increase the
stability and the pharmacokinetics of scFv, Fab or any other
derivative, such as human serum albumin (HSA), CD180, CD200, CD40,
CD20, CD37, CD38, CD148, CD180 and any other antigen specific for B
lymphomas.
The terms "fragments" and "derivatives" of a compound targeting
human CD160 according to the invention are intended to mean
respectively fragments and derivatives which have retained the
binding affinity and the specificity of said compound for human
CD160. Such fragments and derivatives are functional equivalents of
said compound. They bind substantially to the same epitope as said
compound, and/or can compete with said compound for binding to
human CD160, and they retain the binding specificity to human
CD160, which is sufficient for the fragments or derivatives not to
bind to HLA receptors other than human CD160.
The "fragments" and "derivatives" according to the invention have
an affinity similar to the compound of the invention for CD160.
The term "fragment" of a compound targeting human CD160 according
to the invention is intended to mean preferably a format such as a
Fab, a Fab' (reduction of a F(ab')2, for example with
beta-mercaptoethanol), a F(ab')2 or a heavy-chain or light-chain
fragment. The fragments targeting human CD160 according to the
invention comprise at least one heavy chain variable domain (VH)
and/or one light chain variable domain (VL) as defined above.
In one particular implementation of the invention, the compound is
a fragment comprising a light chain (VL) defined by SEQ ID No: 57
and a heavy chain comprising a sequence chosen from SEQ ID No: 36,
SEQ ID No: 37 and SEQ ID No: 38.
The term "derivative" of a compound targeting human CD160 according
to the invention is intended to mean a format of this compound
comprising at least one heavy chain variable domain (VH) and/or one
light chain variable domain (VL), fused to at least one sequence
that is different from the natural sequence (for example a linker
such as SEQ ID No: 39 or a sequence of another protein, in
particular a receptor or a fragment thereof). Said derivative has a
binding affinity to human CD160 that is comparable to that of the
whole compound according to the invention, and also a binding
specificity to human CD160 that is comparable to that of said
compound. In the context of the invention, the term "comparable"
means that the binding affinity or binding specificity can vary
within a limit of 25%. The derivatives can be obtained according to
the general knowledge of those skilled in the art, by enzymatic
reaction, synthesis and/or genetic engineering.
In one particular implementation of the invention, the compound is
a fragment comprising a light chain (VL) defined by SEQ ID No: 57
and a heavy chain comprising a sequence chosen from SEQ ID No: 40
or SEQ ID No: 41.
A derivative according to the invention can be monovalent (a single
site for binding to an antigen, in the case in point human CD160)
or multivalent (at least 2 sites for binding to an antigen or to
several antigens, including at least human CD160). Preferred
multivalent derivatives include bivalent, trivalent and tetravalent
derivatives.
In one embodiment of the invention, the derivative according to the
invention is a multispecific or multifunctional compound, for
example a bispecific antibody (bsab) or a similar molecule, the
epitopes of which can be on identical or different targets. In one
embodiment, the bispecific antibodies can bind to two different
epitopes of CD160. In another embodiment, the bispecific antibodies
can bind to an epitope of CD160 and an epitope of an antigen other
than CD160. The epitopes of interest have been described above in
the present description.
The "single-stranded Fv" or "scFv" antibody derivatives comprise
the VH and VL domains of the antibody, these domains being present
in a single polypeptide chain.
Another derivative according to the invention is a monospecific
multivalent scFv, which can be obtained by binding at least two
monovalent derivatives to one another. The binding may be covalent
or non-covalent. The presence of several CD160-binding sites
increases the binding capacity to this antigen.
Another derivative according to the invention is a multispecific
multivalent scFv.
Among the other derivatives, mention may be made of "diabodies"
which denote small antibody derivatives with two antigen-binding
sites, said fragments comprising a heavy chain variable domain (VH)
linked to a light chain variable domain (VL) in the same
polypeptide chain (VH and VL). The multivalent scFvs are preferably
chosen from diabodies (which are bivalent and are composed of 2
scFvs), triabodies (which are trivalent and are composed of 3
scFvs) and tetrameric scFvs.
Another multivalent derivative according to the invention is a
dimer, each monomer comprising an scFv bonded to a heavy chain
fragment, for example to a CH3 fragment; this corresponds to a
minibody. The 2 scFvs present in the minibody can be identical (the
minibody is then monospecific, since it binds only to human CD160)
or different (the minibody is then bispecific, since it binds, on
the one hand, to human CD160, but also to another antigen).
Another multivalent derivative according to the invention is also a
dimer, each monomer comprising an scFv bonded to heavy chain
fragments, for example to the CH2 and CH3 fragments. Once again,
the 2 scFvs present may be identical or different. In the latter
case, they are referred to as bispecific antibodies.
Another multivalent derivative according to the invention is an
antibody fragment consisting of a single monomeric heavy chain
variable domain. This corresponds to a single-domain antibody (VHH
or sdAb, called Nanobody by Ablynx).
As an example of tetravalent monospecific anti-CD160 derivatives,
mention may also be made of the anti-CD160 molecule in which,
upstream of each variable region of the heavy chain, the VH and CH1
regions are duplicated as described in SEQ ID No: 42 in example 3.
It is then possible to co-express, in mammalian cells, the genes
encoding SEQ ID No: 42 and the anti-CD160 light chain defined by
SEQ ID No: 57 in order to obtain a functional version of functional
monospecific tetravalent anti-CD160.
Another derivative according to the invention is obtained by
recombinantly generating an IgM using one of the chimeric murine
light chain/heavy chain binding human CD160 pairs, according to the
invention.
In another embodiment, a subject of the invention is a composition
comprising at least one compound according to the invention. In one
particular implementation, the composition comprises at least one
compound according to the invention and at least one antibody other
than the compound according to the invention.
In one implementation of the invention, the compound or the
composition as described above is used as a medicament.
The compound targeting human CD160 according to the invention, a
fragment thereof and/or a derivative thereof can be present in a
pharmaceutical composition or a medicament. This pharmaceutical
composition preferably comprises a pharmaceutically acceptable
carrier. The term "pharmaceutically acceptable" refers to a
non-toxic material which is compatible with a biological system
such as a cell, a cell culture, a tissue or an organism, and which
does not interfere with the efficacy of the biological activity of
the active ingredients of the composition. The characteristics of
the carrier will depend on the method of administration.
The pharmaceutical composition or medicament can be in any form
that can be administered to a patient, and includes in particular
solutions, suspensions, lyophilized powders, capsules and
tablets.
The pharmaceutical composition or medicament can be in a form that
is compatible with an injection, i.e. a local injection, i.e. an
intravitreal injection, an administration through the mucosa, an
inhalation, an oral administration and more generally any
formulation suitable for the intended purpose.
A subject of the present invention is also a product comprising a
compound as described in the present application and an antibody
which binds specifically to at least one other antigen which may be
identical to or different from CD160 (in particular to one of the
epitopes of the antigens described above), for simultaneous,
separate or sequential use in the treatment and/or prevention of a
pathological condition which causes a neovascularization, in
particular chosen from neovascular ocular pathological conditions,
primary diabetic retinopathy or age-related macular degeneration
(ARMD), diabetes, diabetic blindness, rheumatoid arthritis,
pre-eclampsia, eclampsia or cancers.
The term "preventing a pathological condition" is intended to mean
the prevention of the occurrence of this disease in a subject, in
particular a human being, in whom the disease has not yet
appeared.
The term "treating a pathological condition" is intended to mean
the inhibition of this disease, i.e. the arrest of its development,
its regression, or the disappearance of the symptoms and
consequences of the disease, or else the disappearance of the
causes of the disease.
More preferentially, the compound according to the invention or the
composition according to the invention is used as an
anti-angiogenic, immunomodulator and/or cytotoxic agent.
A subject of the invention is more particularly a compound
according to the invention, for use thereof as an
anti-angiogenic.
In the context of the present invention, an "anti-angiogenic agent"
or "angiogenesis inhibitor" denotes a compound which inhibits
angiogenesis, vasculogenesis, or else unwanted vascular
permeability, either directly or indirectly.
Preferably, the compound according to the invention can be used for
preventing and/or treating neovascular pathological conditions,
preferably neovascular ocular pathological conditions, diabetes,
diabetic blindness, primary diabetic retinopathy or age-related
macular degeneration, rheumatoid arthritis, pre-eclampsia,
eclampsia or cancers.
The term "neovascular ocular pathological conditions" is intended
to mean all the neovascular ocular diseases or disorders. Several
ocular disorders are associated with pathological angiogenesis. For
example, the development of ARMD is associated with a process
called choroidal neovascularization (CNV). Diabetic macular edema
(DME) is another ocular disorder with an angiogenic component. DME
is the most widespread cause of moderate sight loss in patients
suffering from diabetes and is a common complication of diabetic
retinopathy, a disease affecting the blood cells of the retina.
Another ocular disorder associated with abnormal angiogenesis is
central retinal vein occlusion (CRVO). CRVO is caused by the
obstruction of the central retinal vein which results in an
accumulation of blood and fluid in the retina. The retina can also
become ischaemic, leading to the growth of inappropriate new blood
vessels that can cause additional sight loss and more serious
complications.
Mention may also be made, but non-exhaustively, of other
neovascular ocular pathological conditions, in particular chosen
from Norrie disease; all forms of choroidal neovascularizations,
polypoidal retinochoroidal vasculopathies, retrofoveolar choroidal
neovessels associated with myopia and Sorsby's dystrophia; uveal
melanomas; and rubeosis iridis and neovascular glaucoma, retinal
angiomatous proliferation (RAP), neovascularizations occurring
following corneal transplant complications and/or corneal
infections and/or corneal attacks by the environment, chosen from
pathogenic infections and chemical burns; or all forms of
retinopathies, including diabetic and oedematous ischaemias,
premature diabetic retinopathy, proliferative and non-proliferative
forms of retinopathies, cystoid macular edema, all forms of
age-related macular degeneration (ARMD), in particular the wet
form, all vitelliform macular degenerations, including Best's
disease; ocular angiomas such as Von Hippel-Lindau disease; Eales
disease; Coast disease.
The term "diabetes" is intended to mean any type of diabetes, in
particular sugar diabetes (in relation to insulin) and diabetes
insipidus (in relation to antidiuretic hormone). Among the forms of
sugar diabetes, mention may be made of type 1 diabetes (insulin
dependent), type 2 diabetes (decrease in insulin sensitivity),
gestational diabetes or neonatal diabetes. Among the forms of
diabetes insipidus, mention may be made of central diabetes, due to
a low synthesis of antidiuretic hormone by the pituitary gland, or
peripheral diabetes, due to a low sensitivity of the kidney to
antidiuretic hormone.
The term "cancers" is intended to mean any abnormal proliferation
of cells. The cancers are in particular chosen from breast cancers,
colorectal cancer, bladder cancer, lung cancer and prostate
cancer.
An anti-angiogenic compound of the invention could be used in
particular in the treatment of cancers where the neovascularization
component is an important vector of the propagation of the disease.
Mention is in particular made of breast cancer, colorectal cancer,
non-small-cell lung cancer, non-Hodgkin's lymphoma, urogenital
tumours, such as kidney cancer, prostate cancer, bladder cancer or
renal carcinoma, colon cancer, Hodgkin's lymphoma, liver cancer,
cervical cancer, melanoma, ovarian cancer, mesothelioma and
glioblastoma.
In one particular implementation, the compound of the invention can
be used as a cytotoxic agent.
A "cytotoxic antibody" or "cytotoxic agent" or "anti-tumour agent"
denotes a therapeutic monoclonal antibody (mAb), or a fragment or a
derivative thereof, which induces antibody-dependent effector
cell-mediated cytotoxicity (ADCC), or else complement-dependent
cytotoxicity, or else antibody-dependent cell-mediated phagocytosis
(ADCP) and the direct induction of apoptosis in tumour cells.
Another derivative according to the invention is a compound with
improved cytotoxic activity. A compound with improved cytotoxic
activity can be obtained by grafting the variable chains of
anti-CD160s onto IgG formats with optimized glycosylations of Fc
regions (for example a defucosylation) or by modifying by
engineering the amino acid sequence of the Fc of the antibody of
interest, for example by introducing the DLE triple mutant
(S293D/A330L/I332E). Such a compound can also be obtained by
generating a format of the compound according to the Hexabody
format, or else BITE or alternatively BiKE format (with one valency
directed against CD160 and a second valency directed against CD16)
or TriKE format. Examples of these improvements are mentioned in
Example 03 of this invention. It is also possible to create
compounds of the invention in which one or more residues of an
antibody are replaced with cysteine residues and the free thiol
groups can be used so as to create therapeutic agents such as
immunotoxins, radioimmunoconjugates or else ADCs (Antibody-Drug
Conjugates).
In another bivalent bispecific compound of the invention, it is
also possible to use CD160 as a replacement for CD16 as valency in
an antibody which involves NK cells, on the one hand, and a tumour
antigen on the other non-CD160 valency. This is because CD160 is
also an activator receptor expressed on natural NK cells (see
Examples 15 and 16 of this invention). The interaction of this
compound with the CD160 of NK cells would then result in activating
the NK cells and also bringing these effector cells close to their
tumour target.
Another derivative according to the invention is a compound with an
improved systemic half-life in order to improve its cytotoxic
activity.
The compound or the composition according to the invention can be
used for the treatment of haematological cancers or solid tumours.
Examples of cytotoxic compounds according to the invention are
presented in Examples 3 and 4.
It is known that CD160 is an antigen specific for certain tumour
cells, in particular in the majority of B-cell chronic lymphoid
leukaemias (B-CLL and also hairy cell leukaemias (HCL)), and with a
more heterogeneous expression as a function of patients in cases of
marginal zone lymphomas and in mantle cell lymphomas. However,
CD160 is an antigen which is not at all expressed on normal
circulating B cells. Anti-CD160 antibodies can therefore be used to
specifically kill or inhibit the tumour growth of these B
lymphomas.
The compound of the invention can therefore be used in the
treatment of haematological cancers, in particular chronic lymphoid
leukaemia (CLL), hairy cell leukaemia, acute myeloid leukaemia
(AML), multiple myeloma (MM), or in the treatment of solid tumours,
in particular melanoma, renal carcinoma, lung cancer and in
particular epidermoid lung cancer, neuroblastoma, ovarian
carcinoma, breast cancer, gastric cancer.
The compound of the invention can also be used in the treatment of
haematological cancers in combination with at least one other
antibody, such as anti-CD20 antibodies, in particular rituximab,
ofatumumab, obinutuzumab, ocaratuzumab, or veltuzumab, anti-CD37
antibodies, anti-CD38 antibodies or anti-CD40 antibodies.
The anti-CD160 compound of the invention can be used for modulating
the immunomodulatory activity of CD160 on the NK and T cells of
immunity in the treatment of cancers that respond favourably to
immune checkpoint inhibitors, in particular melanoma,
non-small-cell lung cancer, urogenital tumours such as bladder
cancers or renal carcinoma, colon cancer, Hodgkin's lymphoma, or
breast cancer.
The term "immunomodulatory CD160 activity" denotes one or more
immunoregulatory activities associated with CD160.
The terms "modulate" and "immunomodulator" and their related terms
refer to a reduction or to an increase in the activity of CD160
associated with an upregulation of T-lymphocyte or NK-cell
responses due to its interaction with an anti-CD160 antibody, where
the increase is relative to the activity of CD160 in the absence of
the same antibody. A reduction or an increase in activity is
preferably by at least approximately 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or more. When the CD160 activity is reduced, the
terms "modulator" and "modulate" are interchangeable with the terms
"inhibitor" and "inhibit". When the CD160 activity is increased,
the terms "modulator" and "modulate" are interchangeable with the
terms "activator" and "activate".
The activity of CD160 can be determined quantitatively using
measurements of NK activity (by assaying a marker such as CD69) or
of secretion of cytokines such as IFN gamma as described
respectively in Examples 14 and 15. The activity of CD160 can also
be determined using assessment of T-cell activity by measurement of
proliferation, of cytokine secretion or of activation marker as
CD69 as described in example 19.
The combination of immunomodulators will be key for improving the
clinical responses to immune checkpoint inhibitors.
Thus, in one particular embodiment, the compounds of the invention
are combined with one of these immunomodulators, preferentially
with an anti-PD-1, an anti-CTLA-4 or an anti-PD-L1, in a
composition, where said composition is used as an
immunomodulator.
In another particular embodiment of the invention, the anti-CD160
compound is used as an immunomodulator for the treatment of
bacterial infections for stimulating the defense against pathogenic
bacteria which infect the intestines (in particular Escherichia
coli, Clostridium difficile) or the lungs (in particular
Streptococcus pneumoniae) by activating intra-epithelial innate
lymphoid cells expressing CD160.
A subject of the invention is also a nucleic acid encoding a
compound according to the invention, or a fragment thereof or a
derivative thereof. The term "nucleic acid" is intended to mean a
DNA, cDNA or RNA sequence.
Another subject of the invention is an expression vector comprising
said nucleic acid, or an expression cassette comprising said
nucleic acid. According to the invention, the appropriate
expression vectors can comprise at least one expression control
element functionally linked to the nucleic acid. The expression
control elements are inserted into the vector and make it possible
to regulate the expression of the nucleic acid.
Another subject of the invention is a recombinant cell comprising
an expression vector as described above, or one or more nucleic
acid(s) as described above. According to the invention, examples of
cells that can be used are eukaryotic cells, such as animal, plant,
insect and yeast cells; and prokaryotic cells, such as E. coli. The
means by which the vector carrying the gene can be introduced into
the cells comprise in particular microinjection, electroporation,
transduction or transfection using DEAE-dextran, lipofection,
calcium phosphate or other procedures known to those skilled in the
art. In one preferred embodiment, the eukaryotic expression vectors
which function in eukaryotic cells are used.
Such vectors and nucleic acids can be used in gene or cell therapy,
in order to cause the protein of interest to be produced, in the
case in point the compound according to the invention, by the host
organism.
A subject of the present invention is also a method for treating a
subject, preferably a human being, in which a therapeutically
effective amount of a compound according to the invention is
administered to said subject. The compound according to the
invention is thus administered in a therapeutically effective
amount. A therapeutically effective amount corresponds to an amount
that is sufficient to prevent and/or treat the targeted neovascular
pathological condition. This amount can vary with age, sex of the
subject and stage of the disease and will be determined by those
skilled in the art. A therapeutically effective amount can vary
between 0.01 mg/kg and 50 mg/kg, preferably between 0.1 mg/kg and
20 mg/kg, and more preferably between 0.1 mg/kg and 2 mg/kg, in one
or more daily administrations, for one or more days.
The method of administration can be by injection or by gradual
infusion. The injection can be intravenous, intraperitoneal,
intramuscular, subcutaneous, transdermal, subconjunctival,
intraocular or intravitreal. For a subconjunctival or intravitreal
injection, the therapeutically effective amount of the compound
according to the invention can be between 0.1 mg and 10 mg.
The preparations for parenteral administration can include sterile
aqueous or non-aqueous solutions, suspensions or emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils, such as olive oil, or injectable organic
esters such as ethyl oleate. Aqueous carriers comprise water,
alcohol/water solutions, emulsions or suspensions. The preparations
for parenteral administration can also include sugars and/or
salts.
The compound according to the invention can be labelled. Examples
of labels include toxins, enzymes, radioisotopes, fluorescent
compounds, colloid materials, chemiluminescent compounds, and
bioluminescent compounds. The methods for bonding a label to an
antibody are well known to those skilled in the art.
Another labelling technique consists in coupling the antibody to
low-molecular-weight haptens, it being possible for these haptens
to be specifically modified by means of a second reaction. Examples
of haptens are biotin, which react with avidin, or dinitrophenol,
pyridoxal or fluorescein, which can react with anti-hapten specific
antibodies.
The invention is now illustrated by the following examples and the
attached figures.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1:
A) Binding to CHO-hCD160 compared with CHO WT of the H7 candidates
and of variants thereof in the IgG1 format and IgG1 E345K
B) Binding to CHO-hCD160 compared with CHO WT of the H7 candidates
and of variants thereof in the IgG4 format and IgG4 H310A-H435Q
C) Binding to YT2C2 (NK cell line) of the H7 candidate IgG4
(ELB01101) and of H7 D12 variant in the IgG4 format (ELB01103) with
additional FcRn null mutations (ELB01104), on Fab format (ELB01122)
and on Fab-linker-Fab format (ELB01132)) Black circle: human IgG4,
black triangle: ELB01101 (H7 IgG4), black inverted triangle:
ELB01103, black square: ELB01104, black diamond: ELB01122, black
star: ELB01132.
FIG. 2: Evaluation of the efficacy of an anti-CD160 antibody in the
IgG4 format (ELB01101) over time in comparison with Aflibercept
(Eylea.RTM.) in a model of corneal neovascularization in the rat.
Mean and Error standard are reported on this bargraph. White bar %
vascularisation at Day 8 and black bar % vascularisation at Day
12.
FIG. 3: Mean serum concentration of H7 and H7 variants in different
IgG formats in function of time following two routes of
administration, intravitreal (FIG. 3A) vs intravenous route (FIG.
3B), in rabbit compared to parental CL1-R2, bevacizumab and
ranibizumab.
FIG. 4: Incidence of the anti-CD160 H7 IgG4 (ELB01101) on the total
number of clinically relevant lesions by occasion (grades 3 and 4)
in a monkey model of laser-induced choroidal neovascularization
(combined score of grades 3 and 4/108 laser impacts). The total
number of laser-induced lesions was 108 (corresponding to 12 eyes
with 9 impacts per eye) for the animals treated with the anti-CD160
(H7 IgG4 ELB 01101) or with the carrier control.
FIG. 5: Impact of the anti-CD160 H7 (ELB01101) on scar wound
healing of laser-induced lesions in a monkey model of choroidal
neovascularization. The state of healing and the opening of the
lesions were individually estimated by immunohistochemical analysis
after labelling with an antibody directed against von Willebrand
factor. The percentage (%) of spots during healing (that is to say
covered with the RPE membrane) in comparison with the percentage
(%) of open spots with or without healing is represented as
bargraphs. The % of Open spots with central choroid scar is
represented as plain black bars, % of open spots without central
choroid scar is represented as black diagonal bars, and % of in
healing spots (Spots covered with RPE scar) is represented as white
pigmented bars on the graph.
FIG. 6: Summary of dose efficacy data of H7 D12 in IgG4 format
(ELB01103) in the monkey laser induced ChNV model.
In panel A is reported the impact/incidence of ELB01103 over time
(between Day 0 & Day 14 and Day 0 & Day 28) on % of
clinically relevant lesions (grade 3+4)/Total Number of spots. The
efficacy data at Day 14 and Day 28 are respectively represented as
plain black bar and as diagonal black bar. The mean efficacy level
of anti VEGF as reported in literature in this model is indicated
by a black arrow.
In panel B is reported the impact of ELB01103 on evolution of the
leakage severity over time. The evolution of the leakage severity
is seen by change over time (Day 14 to Day 28) of grade score of
individual clinically relevant ChNV lesion.
In panel C, the impact of dose escalating (0.3 to 3 mg) of ELB01103
on mean change of ChNV area of clinically relevant lesions from Day
14 to Day 28 is described.
In panel D, the impact of a dose of ELB01103 (1 mg) on mean change
of retinal thickness of several types of lesions between Day 14 and
Day 28 is described. In grey is reported the impact on all lesions
(grade 1+2+3+4), in diagonal grey bars is reported the impact on
clinically relevant lesions (grade 3+4) and in black is reported
the impact when only grade 4 lesions are considered.
FIG. 7: Summary of dose efficacy data of H7 D12 in the Fab linker
Fab format (ELB01132) in the monkey laser induced ChNV model.
In panel A is reported the impact/incidence of ELB01132 over time
(between Day 0 & Day 14 and Day 0 & Day 28) on % of
clinically relevant lesions (grade 3+4)/Total Number of spots. The
efficacy data at Day 14 and Day 28 are respectively represented as
plain black bar and as diagonal black bar. The mean efficacy level
of anti VEGF as reported in literature in this model is indicated
by a black arrow.
In panel B is reported the impact of ELB01132 on evolution of the
leakage severity over time. The evolution of the leakage severity
is seen by change over time (Day 14 to Day 28) of grade score of
individual clinically relevant ChNV lesion.
In panel C, the impact of dose escalating (0.25 to 2 mg) of
ELB01132 on mean change of ChNV area of clinically relevant lesions
from Day 14 to Day 28 is described.
In panel D, the impact of a dose of ELB01132 (0.6 mg) on mean
change of retinal thickness of several types of lesions between Day
14 and Day 28 is described. In grey is reported the impact on all
lesions (grade 1+2+3+4), in diagonal grey bars is reported the
impact on clinically relevant lesions (grade 3+4) and in black is
reported the impact when only grade 4 lesions are considered.
FIG. 8: The anti-CD160 H7 antibody in the IgG1 format recognizes
the CD160-positive tumour cells of CLL patients.
The PBMCs isolated from 7 CLL patients were labelled with the
antibodies CL1-R2 (murine anti-CD160 IgG1), anti-CD160 H7 according
to the invention in IgG1 format, or BY55 (murine anti-CD160 IgM),
in a CD19/CD5/CD3/CD56 panel. The CD5+CD19+ tumour cells were
analysed in order to measure the fluorescence intensity of the
CD160 labelling. CD160 expression is detectable on all the CLL
samples with variable intensities. The H7 IgG1 antibody binds
efficiently to the tumour cells in 6/7 of the CLL samples
examined.
auto=autofluorescence of the cells; isotype=IgG1 or IgM, murine,
irrelevant, negative control
FIG. 9: The anti-CD160 H7 antibody in the IgG1 format kills the
cells expressing CD160 via an ADCC mechanism.
NK cells purified from the blood of a healthy donor were used as
effectors in a test measuring the ADCC activity of the anti-CD160
H7 IgG1 antibody. The E300-CD160 target cells (transfected pre-B
human cell line expressing CD160) were labelled with CFSE, and
incubated with the effector NK cells in the presence of the H7 IgG1
antibody or of a human IgG1 isotype control, at the effector/target
ratios indicated (1/1, 1/5 and 1/10). The percentages of target
cells killed were measured by labelling with 7AAD and flow
cytometry analysis. The percentages of doubly labelled 7AAD+CFSE+
dead cells are indicated in the top right quadrant on the dot-plots
presented.
FIG. 10: The anti-CD160 H7 antibody in the IgG1 format activates NK
cells.
A) The H7 IgG1 antibody binds to human NK cells. NK cells were
purified from the blood of a healthy donor using a Miltenyi kit
(ref. 130-092-657) and an autoMACS.TM. (Miltenyi ref. 130-092-545).
After saturation of the cell surface Fc receptors with human IgG Fc
fragment (Rockland ref. 009-0103) for 15 min, 5.times.10.sup.5 NK
were incubated for 20 min at 4.degree. C. with 0.25 .mu.g of H7
IgG1 antibody or of a human IgG1 (isotype control), coupled to
phycoerythrin using an antibody conjugation kit (Lynx ref. PE
LNK021RPE) and a CD56-APC antibody. The histograms show the
fluorescence profiles obtained with H7 IgG.sub.1 (black) or the
hIgG.sub.1 control (grey), analysed on the CD56-positive
population.
B) H7 IgG.sub.1 induces the production of interferon-gamma
(IFN-.gamma.) by NK cells. NK cells purified from the blood of a
healthy donor were cultured for 24 h in wells of 96-well plates
(1.times.10.sup.+6 cells per well) alone or in the presence of the
H7 IgG.sub.1 antibody, or of a human IgG1 isotype control,
concentrated to 1 or 10 .mu.g/ml. The IFN-.gamma. was assayed by
ELISA in the culture supernatants. The results presented are means
of triplicates+/-sem.
C) H7 IgG.sub.1 induces the expression of the activation marker
CD69 on NK cells. In the same experiment, the NK cells were
collected after 24 h of culture and labelled with an anti-CD69
antibody conjugated to the fluorochrome APC. The percentages of
CD69-positive cells were analysed by flow cytometry. The
means(+/-sem) were calculated from triplicates.
FIG. 11: The anti-CD160 H7 antibody in the IgG1 format, but not
IgG4, activates NK cells.
NK cells purified from the blood of a healthy donor were cultured
alone or in the presence of the following antibodies concentrated
to 5 .mu.g/ml: H7 IgG1, H7 IgG4, their respective human IgG1 or
IgG4 isotype controls, or the antibodies ELB01103, ELB01104 and
ELB01106, which are variants derived from the H7 antibody in the
IgG4 format. The anti-CD16 antibody (Ebioscience cat #16-0166) is
used as positive control. The NK cells (5.times.10.sup.+5 per well)
were collected after 24 h of culture and labelled with an anti-CD69
antibody conjugated to the fluorochrome APC. The percentages of
CD69-positive cells were analysed by flow cytometry (means of
triplicates+/-SD). The anti-CD160 H7 in the IgG1 format induces the
expression of the activation marker CD69 on NK cells, whereas the
same antibody in the IgG4 format has no effect. The H7 variants
IgG4 (ELB01103, ELB01104 and ELB01106) also do not show any
activating effect on NK cells.
FIG. 12: The variants derived from the anti-CD160 H7 antibody in
the IgG1 and E345K/IgG1 formats have an increased capacity to
activate NK cells.
NK cells purified from the blood of a healthy donor were cultured
for 24 h in wells of 96-well plates (1.times.10.sup.+6 cells per
well), alone or in the presence of the anti-CD160 H7 IgG1 antibody,
or of the variants ELB02102, ELB02103, ELB02104 (all three in the
IgG1 format), ELB02112, ELB02113 or ELB02114 (all three in the
E345K/IgG1 format) produced by ElsaLys, at doses of 0.001 to 10
.mu.g/ml. A human IgG1 at 10 .mu.g/ml was used as negative isotype
control, and an anti-CD16 (Ebioscience cat #16-0166) was used as
positive control.
A) The IFN-.gamma. was assayed by ELISA in the culture
supernatants. The results presented are means of
triplicates+/-sem.
B) The NK cells were collected and labelled with an anti-CD69
antibody conjugated to the fluorochrome APC. The percentages of
CD69-positive cells were analysed by flow cytometry. The
means(+/-sem) were calculated from triplicates.
All of these results show that the three H7 variants in the IgG1
format (ELB02102, ELB02103, ELB02104) are much more potent than the
original H7 IgG1 antibody in terms of activating NK cells, with an
improvement of 2 to 3 logs of the EC50 values.
The three H7 variants in the E345K/IgG1 format show a further
increased capacity for inducing IFN-.gamma. production, with an
additional improvement of 2 logs in the EC50 values (4 logs
compared with the original H7 IgG1 antibody).
FIG. 13: The variants derived from the anti-CD160 H7 antibody in
the IgG1 and E345K/IgG1 formats efficiently label NK (FIG. 13A) and
CD8+T (FIG. 13B) cells.
The PBMCs (peripheral blood mononuclear cells) from two healthy
donors were analyzed by flow cytometry after immunolabelling with
anti-CD45, CD3, CD4, CD8 and CD19 antibodies and with the
anti-CD160 antibodies indicated (0.25 .mu.g for 5.times.10.sup.+5
PBMCs). An irrelevant human IgG1 (hIgG1) was used as negative
control. Non-hatched bars: donor 1; hatched bars: donor 2.
FIG. 14: Binding to CHO-CD160 TM (transmembrane), CHO-CD160 GPI
(glycosylphosphatidylinositol) and CHO of CL1R2, ELB02101 (H7 IgG1)
candidates and variants ELB02104, ELB02114 and ELB01103.
Humanized ELB02101 (H7 IgG1) and the variants ELB02104, ELB02114
and ELB01103 bind unexpectedly to human CD160-TM expressed
recombinantly by the CHO-S cells while parental CL1R2 mAb doesn't
bind. Black bars: CHO, hatched bars: CHO-CD160-GPI
(glycosylphosphatidylinositol), checkerboard bars: CHO-CD160TM
(transmembrane)
FIG. 15: T CD4 re-activation through the blocking of HVEM-CD160
interaction by the H7 A09 variant in IgG1 format (ELB02104).
T CD4 lymphocytes cells purified from the blood of a healthy donor
were cultured for 16 h in 96-well plates (1.times.10.sup.+6 cells
per well), in the presence of the anti-CD160 mAb: ELB02104 or with
the appropriate control isotype at 10 .mu.g/ml and with anti-CD3
(clone UTCH1) mAb+/-anti-CD28 (clone CD28.2) mAb+/-HVEM protein
(10334-H08H, Sino biological) coated on the plate. T CD4
lymphocytes were collected and labelled with a viability marker:
Zombie NIR, an anti-CD45RA antibody conjugated to the fluorochrome
BB515 targeting Naive/Memory cells, with an anti-CD160 (clone BY55)
antibody conjugated to the fluorochrome Alexa fluor 647 targeting
CD160 expressing cells and with an anti-CD69 antibody conjugated to
the fluorochrome PE targeting activated cells. The percentage of
Zombie NIR-/CD45RA.sup.high+/CD160+/CD69+ positive cells were
analyzed by flow cytometry. The means(+/-sem) were calculated from
duplicates. ELB02104 blocks HVEM-CD160 interaction and removes the
inhibition of TCD4 cells induced by HVEM protein as shown by the
upregulation of CD69, an activation marker expressed by T CD4
CD45RA.sup.High CD160+ cells. White bars: human IgG1 control
isotype, black bars: ELB02104
EXAMPLE 1
Study of the Binding of the Antibodies According to the
Invention
The determination and the comparison of the affinities described in
Table 1 of the murine anti-hCD160 CL1-R2 or in its derived forms
(chimeric IgG1 and chimeric IgG4) or of the compounds of the
invention (H7 IgG1, H7 IgG4) were carried out using the principle
of biolayer interferometry on an Octet K2 instrument (Pall
ForteBio) equipped with fibre optic biosensors of various types
depending on the experiments. The capacity of the antibodies
according to the invention to bind to their target was studied by
measuring the human CD160 protein/antibody interaction.
For this, the monomeric anti-human CD160 antibodies of high purity
(purified on protein A and then by gel filtration) were prepared by
techniques well known to those skilled in the art. The protein
region corresponding to the soluble form of the recombinant human
CD160 protein bearing a C-terminal tag of 6 histidine residues
(from R&D SYSTEMS) is used in its commercial formulation.
The affinities of the various anti-CD160 candidates to be tested,
i.e. the compounds of the invention, were compared to that of the
chimeric antibodies and of CL1-R2.
All the experiments were carried out at 30.degree. C. in the
running buffer recommended by Fortebio (PBS with 0.1% (p/v) of
bovine serum albumin (BSA) and 0.02% (v/v) Tween-20). This buffer
was also used for diluting the various ligands and analytes. The
samples deposited in a 96-well microplate (cat #738-0026, Dutscher)
were shaken at 1000 revolutions per minute.
The CD160 protein comprising a 6-histidine-residue tag which is
biotinylated is used as ligand on streptavidin biosensor and the
compounds, according to the invention, anti-hCD160 (IgG1 and IgG4
formats) and anti-CD160 and chimeric compounds are used as
analytes.
This hCD160-his protein was biotinylated using the EZ-Link
sulfo-NHS-LC-biotin method (Thermo Fisher Scientific) according to
the supplier's recommendations and validated for its homogeneity,
the absence of aggregates and its capacity to be recognized by
anti-CD160s just as well as the non-biotinylated protein.
Immobilization tests with various protein concentrations showed
that a concentration of 10 nM was optimal. The biotinylated CD160
protein was therefore immobilized at a concentration of 0.3
.mu.g/ml (i.e. 10 nM) on streptavidin biosensors for 10 min. A
typical immobilization results in a signal of 2+/-0.3 nm.
The kinetic constants (K.sub.D, k.sub.on and k.sub.off also called
K.sub.dis) were determined for each of the purified antibodies
(molecular weight 150 kDa) by addition of 6 concentrations of
antibodies (of 3.125, 6.25, 12.5, 25, 50 and 100 nM). Between the
measurements, the surfaces of the biosensor were regenerated by
exposing them to 3 cycles of 5 sec in 10 mM glycine, pH 2, followed
by 5 sec in running buffer. The association and dissociation phases
were measured for 300 seconds. All the measurements were corrected
for the basic drift by subtracting a reference well with a ligand
subjected only to the running buffer.
The dissociation constants and the association (k.sub.on) and
dissociation (k.sub.dis) rate constants for each antibody were
calculated by applying a 1:1 interaction model, with overall
mathematical modelling of curves (fit) (Rmax bound by the sensor)
on the ForteBio 9.0 data analysis software. The curves that could
not be reliably modelled using the software (most of the time with
an R2 full <0.925), generally caused by binding according to a
heterogeneous mode, were excluded from the analyses.
For each anti-CD160, the dissociation constants (KD), and the
association (k.sub.on) and dissociation (k.sub.dis) rate constants,
and also the binding response were compared for anti-CD160 antibody
concentrations of 50 nM and are reported in Table 1.
TABLE-US-00001 TABLE 1 Measurement by Biolayer interferometry (BLI)
of the affinity of the recombinant human CD160/anti-hCD160
interaction for the murine CL1-R2 antibody, the chimerized
antibodies issued from CL1-R2 (in the human IgG1 (chIgG1) or IgG4
(chIgG4) formats) and the H7 antibody of the invention (in the
human IgG1 (H7 IgG1) or IgG4 (H7 IgG4) format). Binding response,
at 50 nM K.sub.D gain of anti- Anti- K.sub.D k.sub.on k.sub.dis
(/K.sub.D CD160 hCD160 K.sub.D Error k.sub.on Error k.sub.dis Error
parental (arbitrary (batch) (nM) (*1E-.sup.11) (1E.sup.+5/Ms)
(*1E.sup.+03) (1E.sup.-03/s) (*1- E.sup.-05) CL1-R2) units nm) H7
IgG1 4.00 2.83E-11 5.87 3.06 2.35 1.12 3.75 1.01 chIgG1 14.3
16.5E-11 2.08 1.94 2.97 2.06 1.07 0.41 H7 IgG4 4.49 5.39E-11 3.50
2.65 1.57 1.47 3.34 0.60 chIgG4 14.8 1.52E-10 2.18 1.83 3.23 1.92
1.04 0.41 CL1-R2 15.4 1.74E-10 1.83 1.69 2.83 1.83 1 0.60
The affinity measurements clearly show here, unexpectedly, that the
H7, in the IgG1 and IgG4 formats, has a much better affinity for
human CD160 than the murine CL1-R2 and its respective chimeric
human IgG1 and IgG4 forms. The gain in K.sub.D (cf. K.sub.D gain,
Table 1, column 8) compared to the parental CL1-R2 anti-CD160
K.sub.D is about 3.75 and 3.34 for H7 in the human IgG1 and IgG4
format respectively. For one and the same concentration of 50 nM of
antibody, a better response for the H7 IgG1 than for H7 IgG4 and
CL1-R2 and a worse response for the two anti-CD160s in the chimeric
formats were also obtained (Table 1, column 9).
It was also verified that the H7 bind well to their target, by flow
cytometry study on recombinant E300-hCD160 cells overexpressing
human CD160 and by ELISA on CD160 protein and on peptide of a
protein sequence necessary and sufficient for there to be binding
of the anti-hCD160 antibody, identified by peptide scanning of the
human CD160 sequence.
The H7 candidate is the candidate chosen for the remainder of the
experiments, in particular for being affinity matured and for being
derived into various formats of IgG or of IgG fragments suitable
for the various clinical indications targeted.
EXAMPLE 2
The Variants of the H7 Antibody
Binding Profile, by ELISA, FACS and SPR, of a Panel of H7 Variants
in the Form of Phages and Soluble Fabs Produced in Periplasmic
Extracts of Bacteria.
In order to obtain variants derived from the humanized anti-CD160
candidate H7, a site-directed mutagenesis of the residues of the
specific complementarity-determining regions (CDRs) of the heavy
and light chain variable domains (VH and VL respectively) of the H7
antibody were combined with a selection on protein and on CHO cells
overexpressing hCD160 by phage display of the variants in the Fab
format.
Thus, phage clones were generated and also made it possible to
produce periplasmic extracts of bacteria, containing unpurified
soluble Fabs. The individual clones were selected for their binding
capacity to the human CD160 protein, by ELISA, and to cells
expressing human CD160 by FACS, this being i) either in the form of
gene III-Fab fusion proteins expressed at the surface of the
filamentous phage or ii) in the form of periplasmic extracts
containing fragments of unpurified soluble Fabs. The results of the
experiments termed phage ELISA and phage FACS are reported in Table
2. The individual clones (in the form of periplasmic extracts
containing fragments of soluble Fabs) were also classified
according to their kinetic dissociation constant (k.sub.dis).
The summary of the data obtained for the 6 H7 variants and in the
form of periplasmic extracts containing fragments of unpurified
soluble Fabs (PE ELISA and PE FACS) is given in Table 2.
From a practical point of view, the binding of the phage to human
CD160 was detected using an anti-M13 HRP conjugated antibody. The
binding of the phage to the cells expressing CD160 was detected
using a mouse anti-M13-biotin antibody followed by
Streptavidin-PE.
In the phage ELISA, most of the phages were able to bind the human
CD160 protein with high optical density (OD) values at 450 nm (OD
450: 1.0-6.0) and with a degree of binding success of 83% (OD at
450 nm.gtoreq.10 mean of the background noise). It is important to
note that, for the H7 WT Fab phage, the OD 450 values obtained were
as low as 0.06-0.07.
Similar results were obtained from the phage FACS assay, with a
degree of success of 91% (clones with more than 5% of binding to
cells expressing CD160, 3 times the virgin MFI values and no
binding to the CHO-S WT cells was considered positive). As in the
phage ELISA, the binding values obtained for the Fab H7 WT phage
FACS were much lower compared with the other clones.
The binding of the clones selected (from the rounds of selection on
CD160 protein and on CHO-CD160 cells) as soluble Fab was also
looked at by ELISA and FACS using periplasmic extracts (P.E). The
binding of the soluble Fabs to the human CD160 protein, using
ELISA, was detected using an anti-c-myc antibody followed by an
anti-mouse HRP conjugated antibody. The binding of the soluble Fabs
to the CHO cells overexpressing human CD160 was detected using an
anti-c-myc antibody followed by a goat anti-mouse APC conjugated
antibody. The results of the ELISA and FACS experiments using Fabs
expressed at the surface of the phages or in periplasmic extracts
confirmed the binding capacity of each of the affinity-matured H7
clones to human CD160.
TABLE-US-00002 TABLE 2 Class of affinity-matured heavy chain H7
variants in the form of phages and soluble Fabs produced in
periplasmic extracts of bacteria, clone identification, binding
profile by ELISA, FACS and SPR. Phage ELISA P.E ELISA Phage P.E H7
VH O.D.sub.450 nm on O.D.sub.450 nm on FACS FACS Anti-CD160 phage
family biotinylated biotinylated (% (% cloning No. VH CD160 CD160
binding) binding) FJ1516MP02F04 1 SEQ ID No: 25 3.323 0.436 96.53
9.86 FJ1516MP02D09 2 SEQ ID No: 26 3.63 0.174 76.36 1.08
FJ1516MP02A12 3 SEQ ID No: 27 6 0.148 92.06 1.23 FJ1516MP02G05 4
SEQ ID No: 28 3.458 0.141 86.86 1.06 FJ1516MP02D12 5 SEQ ID No: 29
6 0.312 95.09 1.89 FJ1516MP02A09 6 SEQ ID No: 30 6 0.124 94.19 0.54
FJ1516MP02E07* WT 3.209 0.145 70.44 0.21 FJ1516MP02G12** WT 1.007
0.131 11.75 0.26 *WT H7 obtained in the screening **Fab H7 WT
control produced
The amino acid sequences of the various clones originating from
different rounds of selection (FJ1516MP02 and FJ1516MP03) were
extracted using the CLC Main Workbench software. The mutant
V.kappa. and VH sequences were aligned separately with respect to
the reference H7 VH and V.kappa. sequences. All the clones selected
contain a V.kappa. sequence corresponding to the V.kappa. sequence
of the H7 WT. For the heavy chain, starting from 156 valid
sequences, six also corresponded to the VH sequence of H7 WT. All
the other VH sequences contained 2 to 6 mutations (as designed in
the library) relative to WT and were grouped together in 6
different classes (VH variant sequences 1 to 6) (see column 2, VH
family in Table 2 above).
For the remainder of the characterization experiments, a panel
containing a clone representative of each different VH variant was
selected for the production of phage and of soluble Fab in P.E. The
list of representatives selected is given in column 1 of Table 2
and their corresponding VH family in column 2. The clone
representative of class 5 is D12, the clone representative of class
1 is F04 and the clone representative of class 6 is A09. The VH
amino acid sequences of the clones representing the various classes
1 to 6 of the variants of the anti-CD160 H7 are mentioned in Table
2.
The alignment of the protein sequences of the VH regions of these 6
classes of H7 variants showed common constants between the various
classes of variants in terms of the positions of the mutated
residues and in terms of the nature of the mutations
introduced.
SPR Measurement of Interaction with Human CD160 of the 6 Variant
Fabs of H7 Using Soluble Fabs Produced in Bacterial Periplasmic
Extracts
The binding capacity of the H7 variants was also tested by surface
plasmon resonance (SPR). For this, a Biacore 3000 (GE Healthcare)
was used. 50 .mu.g/ml of human CD160 (R & D Systems) in an
acetate buffer, pH 4.5, were immobilized on a CM5 chip (GE
Healthcare) at 1250-2000 resonance units (RUs). The integrity of
the immobilized human CD160 was confirmed using the anti-huCD160 H7
IgG1 antibody. For the kinetic measurements, concentrations in
cascade of human ECD160 with two-fold dilutions (0.15 .mu.M-10
.mu.M) were injected, twice, in PBS, with a Biacore P20 buffer at
25.degree. C. and a flow rate of 30 .mu.l/min.
The regeneration conditions were tested and 10 pi of 10 mM NaOH/1M
NaCl were injected for the regeneration between the sample
injections. To analyse the binding of the clones to human CD160,
the periplasmic extracts containing the soluble Fabs were diluted
to 1:5 in BIACORE P20 buffer (10 mM Hepes, 150 mM NaCl, 3 mM EDTA,
0.005% Tween-20) with a flow rate of 30 .mu.l/min for 120 seconds
for the association; the dissociation was measured for 300
seconds.
TABLE-US-00003 TABLE 3 Absolute response of the binding of the
periplasmic extracts containing soluble Fabs representative of the
various classes of affinity-matured H7 variants Binding response,
Biacore RU arbitrary Biacore R.U ratio H7 VH unit (at t = 120 s R.U
ratio (variant/ Anti-CD160 phage family post-mab (variant/ FaWT
clone No. injection) H7 WT) 2E07) FJ1516MP02F04 1 50.6401 8.3 3.9
FJ1516MP02D09 2 19.2957 3.2 1.5 FJ1516MP02A12 3 20.1003 3.3 1.5
FJ1516MP02G05 4 16.0439 2.6 1.2 FJ1516MP02D12 5 43.7766 7.2 3.3
FJ1516MP02A09 6 16.7186 2.7 1.3 FJ1516MP02E07* WT 13.1271 2.2 1
FJ1516MP02G12** WT 6.08838 1 0.5 *WT H7 obtained by selection in
the screening **Fab H7 WT control produced
The maximum absolute response of the binding of each of these
variants (expressed as measurement of arbitrary response units at
time=120 seconds post-injection of the antibody) was evaluated on a
surface coated with CD160 protein. These data are in Table 3.
It was then possible to calculate the ratio of binding of each
variant to that of the H7 Fab produced as a control or an H7 Fab
isolated on a phage during the screening (cf. Table 3, columns 4
and 5).
The data grouped together in Table 3 demonstrate that the soluble
Fabs are capable of binding human CD160 in agreement with what was
previously observed by ELISA and FACS.
The clones F04, D12 and A09 (variant VH of classes 1, 5 and 6
respectively) showed the highest binding values (RU) and also RU
ratios (cf. column 4, variant RU/WT RU Table 3) that were also
high, of 8.3; 7.2 and 2.7 times, respectively.
EXAMPLE 3
Design and Generation of Various Monospecific Antibody Formats for
the Variants for the Oncology and Ophthalmology Indications
The clones FJ1516MP02F04 or F04 and FJ1516MP02D12 or D12 were
formatted in IgG in order to study whether the mutations present
could make it possible to bind to CD160 with affinities higher than
that of the H7 antibody. The FJ1516MP02A09 or A09 variant is the
only representative which also has an association/dissociation
profile very different from the others and, in this respect, will
also be studied further in the IgG format.
3.1) Protein Sequences of the Anti-CD160 Constructs to be Tested in
Ophthalmology Using H7 and its Variants
Thus, for ophthalmology, the F04 and D12 variants were generated in
formats (IgG4 or IgG1 N297Q for example) chosen to not interact, or
to interact minimally, with Fc receptors (FcR), and/or others for
reducing the systemic half-life of the therapeutic anti-CD160
antibody or fragment, without reducing too much its intravitreal
half-life by producing in this case either mutations that reduce
the systemic half-life of the antibody, or either by proposing
formats of antibody fragments without Fc region.
Reduction of the Systemic Half-Life and of the Engagement of FcRs
and of FcRns by Formatting the H7 Candidate and its Variants in an
IgG4 or IgG1 N297Q+/-FcRn Null Mutations
Thus, for ophthalmology, a first possibility to reduce the systemic
half-life of the therapeutic anti-CD160 antibody, is to format the
F04 and D12 variants by cloning the variable regions on an IgG4
S228P-R409K or IgG1 N297Q structure chosen to not interact, or to
interact minimally, with Fc receptors (FcR). On this backbone it
also possible to insert the mutations S228P/R409K/H310A/H435Q or
I253A in the Fc region of the Mab to reduce its interaction with
human neo natal Fc receptor (FcRn and "FcRn null mutations"
described in (Olafsen, 2012). This could be achieved by different
combinations of heavy and light chain sequences as described in
Table 4.
TABLE-US-00004 TABLE 4 Name of the compounds and VH and VL
sequences ELB code Anti-CD160 name/code Heavy chain Light chain
ELB01101 H7 IgG4 S228P/R409K SEQ ID No: 58 SEQ ID No: 57 ELB01103
D12 IgG4 S228P/R409K SEQ ID No: 60 SEQ ID No: 57 ELB01106 F04 IgG4
S228P/R409K SEQ ID No: 61 SEQ ID No: 57 ELB01111 H7 IgG1 N297Q SEQ
ID No: 59 SEQ ID No: 57 ELB01102 H7 IgG4 S228P/R409K/ SEQ ID No: 62
SEQ ID No: 57 H310A/H435Q ELB01104 D12 IgG4 S228P/R409K/ SEQ ID No:
63 SEQ ID No: 57 H310A/H435Q -- F04 IgG4 S228P/R409K/ SEQ ID No: 10
SEQ ID No: 57 H310A/H435Q -- D12 IgG4 S228P/R409K/ SEQ ID No: 9 SEQ
ID No: 57 I253A -- F04 IgG4 S228P/R409K/ SEQ ID No: 12 SEQ ID No:
57 I253A
SEQ ID No: 57 results from the fusion of the variable region
defined by SEQ ID No: 14 to the constant region defined by SEQ ID
No: 22.
Reduction of the Systemic Half-Life and of the Engagement of FcRs
and of FcRns by Formatting the H7 Candidate and its Variants in
Antibody Fragments.
Another way to reduce the systemic half-life and also the
engagement of FcRs and of FcRns of a therapeutic antibody injected
in IVT is the formatting of the H7 antibody and its variants in
antibody fragments (Fab, Fab'2 for example). Thus, the H7 and its
variants are formatted in Fab format (with the following Fab
constant chain synthesized by genetic engineering and produced in
bacteria or in CHO cells) by combining the light chain of H7 (SEQ
ID No: 57) with one of the following heavy chains in order to
produce the corresponding format (see Table 4). Fab CH1 IgG1
ELB01121 (SEQ ID No: 36) Fab CH1 IgG1 D12 ELB01122 (SEQ ID No:
37)
The Fab'2 format is produced for the D12 variant (SEQ ID No: 38)
(recombinantly or by enzymatic cleavage (Ides fabricator, GeNovis))
with two disulfide bridges instead of one or with or without
leucine zipper.
A Fab linker Fab was generated in which the two sequences of the
heavy chains are linked between the C terminal of the first Fab
with the N-terminal of the second Fab by means of a linker protein
sequence (SEQ ID No: 39), which gives a heavy chain of the
Fab-linker-Fab molecule ELB01131 defined by SEQ ID No: 40 and
ELB01132 defined by SEQ ID No: 41.
A tetravalent format with 4 anti-CD160 D12 Fabs was created using
as heavy chain of the tetravalent the IgG1 N297Q H310A-H435Q D12
(SEQ ID No: 42; ELB012001).
All of the sequences of these heavy chains are the sequences of a
mature heavy chain and the sequence of a signal peptide, such as
one of those described in SEQ ID No: 18 or 19, must be added in the
N-terminal position.
3.2) Protein Sequences of the Anti-CD160 Constructs to be Tested in
Oncology Using H7 and its Variants.
The different formats that were compared for oncology are H7 and
its three variants D12, F04 and A09 in the IgG1 format, and also in
the Hexabody format of Genmab and in the Bite format for D12.
The Hexabody format (Diebolder et al., 2014; de Jong et al., 2016)
was generated so as to optimize the cytotoxicity of the anti-CD160
in order to activate complement and to improve the capacity of the
antibody to induce lysis, by CDC and ADCC, of CD160-positive tumour
cells. Wang et al. (Wang et al., 2016) identified mutations E345K
(SEQ ID No: 43) or E430G (SEQ ID No: 44) which allow the production
of a monomeric Hexabody with improved effector functions (CDC and
ADCC) while at the same time retaining equivalent pharmacokinetics
and pharmaceutical developability. The IgG1 thus mutated
hexamerizes following binding of the antibody to the antigen
expressed by the target cell and this hexamerization improves the
effector functions (CDC and ADCC) of the antibody. The molecular
construction of the D12 and F04 variants in the IgG2a/murine kappa
format, as preclinical tools, was also carried out.
TABLE-US-00005 TABLE 5 Name and ElsaLys codes (ELB) of the
compounds and VH and VL sequences ELB code Anti-CD160 name/code
Heavy chain Light chain ELB02101 Human H7 IgG1 SEQ ID No: 64 SEQ ID
No: 57 ELB02102 D12 in the IgG1 format SEQ ID No: 45 SEQ ID No: 57
ELB02103 F04 in the IgG1 format SEQ ID No: 46 SEQ ID No: 57
ELB02104 A09 in the IgG1 format SEQ ID No: 47 SEQ ID No: 57
ELB02111 H7 in the IgG1 E345K format SEQ ID No: 48 SEQ ID No: 57
ELB02112 D12 in the IgG1 E345K format SEQ ID No: 49 SEQ ID No: 57
ELB02113 F04 in the IgG1 E345K format SEQ ID No: 50 SEQ ID No: 57
ELB02114 A09 in the IgG1 E345K format SEQ ID No: 51 SEQ ID No: 57
ELB02102-02 D12 in the murine IgG2a format SEQ ID No: 53 - SEQ ID
No: 54 ELB02103-02 F04 in the murine IgG2a format SEQ ID No: 55 SEQ
ID No: 56
Moreover, the protein sequence of the D12 variant in the BITE
format, ELB02122, is defined by SEQ ID No: 52.
EXAMPLE 4
Biophysical Characterization of the H7 Variants in the IgG4, IgG1
and IgG1 E345K Formats
4.1) Evaluation of the Impact of the Mutations of the H7 Variants
on the Thermostability of the Anti-CD160s.
Thermostability is a common method used for studying the stability
of a protein. Thermostability results i) from the intrinsic
stability of a protein (propensity to form aggregates) linked to
its three-dimensional structure that results from its primary
sequence, and ii) from the sample storage and formulation
conditions (pH, salts, and components of the sample). According to
the method based on the differential capacity of Sypro Orange
(Thermofischer Scientific, S-6650, batch 1608495) to bind to the
hydrophobic regions of the protein in native or denatured form, the
thermostability of the variants of the anti-hCD160 H7 candidate in
various IgG formats was evaluated.
The samples are tested in quadruplicate in a 96-well PCR plate, in
a final volume of 30 .mu.l at a final concentration of 0.1 mg/ml in
1.times. PBS, 5.times. Sypro Orange. The stock solution of Sypro
Orange (5000.times. stock in 100% DMSO) is prepared at a final
concentration of 10.times. in 1.times. PBS. The plate is then
subjected to a temperature gradient of 22.degree. C. to 99.degree.
C. (over the course of approximately 1 h 30) in an Applied
Biosystems.RTM. 7500 Real-Time PCR system device. The data analysis
(raw data and first derivative giving the Tm for each antibody
domain) was carried out using the software: Protein Thermal Shift
(Thermofischer Scientific). The results are presented in the
following table:
TABLE-US-00006 TABLE 6 Tm results for H7 and the H7 variants in the
IgG4, IgG1 and IgG1 E345K formats Mean Standard Mean Standard Tm1,
deviation Tm2, deviation Code Protein .degree. C. Tm1 .degree. C.
Tm2 ELB01101 H7 IgG4 WT 65.49 0.10 ELB02101 H7 IgG1 WT 68.98 0.12
ELB01103 D12 IgG4 65.02 0.07 72.62 0.07 ELB01106 F04 IgG4 60.66
0.07 69.85 0.07 H310A-H435Q ELB01102 H7 IgG4 60.77 0.04 69.13 0.04
H310A-H435Q ELB01104 D12 IgG4 60.55 0.09 71.61 0.09 H310A-H435Q
ELB02104 A09 IgG1 69.38 0.21 74.75 0.07 ELB02103 F04 IgG1 69.63
0.07 ELB02102 D12 IgG1 69.60 0.07 ELB02113 F04 IgG1 E345K 69.52
0.12 ELB02114 A09 IgG1 E345K 69.05 0.04 74.28 0.00 ELB02112 D12
IgG1 E345K 69.67 0.06 ELB01111 H7 IgG1 N297Q 59.58 0.16 70.50 0.11
ELB01112 H7 IgG1 N297Q 53.96 0.00 70.17 0.04 H310A-H435Q
The analysis of the results of Table 6 shows that the mean Tm of H7
in the IgG1 format (H7 IgG1 WT) is 3.5.degree. C. higher compared
with H7 in the IgG4 format (H7 IgG4 WT). Regarding the H7 variants,
the antibodies have a Tm very close to that of H7.
4.2) BLI Measurement for Comparison of the Affinity for the
Recombinant Human CD160, of H7 and of the Different Variants
Thereof, this Being in Different IgG Formats
The affinities herein were measured as described in Example 2 with
a design where the biotinylated CD160 protein is captured at 10 nM
on a streptavidin biosensor and where the analytes are the
anti-CD160s. The anti-CD160 concentrations tested were 3.13, 6.25,
12.5, 25, 50 and 100 nM and the glycine concentration, pH 2, was 10
mM for each regeneration.
The sensorgrams and the affinities measured for the CD160 protein,
of the anti-CD160 H7 antibodies and of the variants thereof, are
presented in the following tables:
TABLE-US-00007 TABLE 7 Measurement of the affinities for the CD160
protein, of the anti-CD160 H7 antibody and of the D12 variant
thereof in the IgG4 S228P-R409Q format Reference IgG, No. KD (M)
kon(1/Ms) kdis(1/s) RMax Full X{circumflex over ( )}2 Full
R{circumflex over ( )}2 ELB01101 H7 2.61E-09 4.69E+05 1.23E-03
0.4394 0.1146 0.9907 IgG4 ELB01103 1.52E-09 4.94E+05 7 52E-04
0.8059 0.2698 0.9947
TABLE-US-00008 TABLE 8 Measurement of the affinities for the CD160
protein, of the anti-CD160 H7 antibody and of the variants thereof
in the IgG4 S228P-R409Q-H310A-H435Q format Reference IgG, No. KD
(M) kon(1/Ms) kdis(1/s) RMax Full X{circumflex over ( )}2 Full
R{circumflex over ( )}2 ELB01102 H7 2.91E-09 5.07E+05 1.48E-03
0.4832 0.1092 0.9924 IgG4 H310A-H435Q ELB01104 1.49E-09 4.99E+05
7.44E-04 0.7845 0.3143 0.9935 ELB01106 1.63E-09 4.53E+05 7.36E-04
0.7779 0.1797 0.996
TABLE-US-00009 TABLE 9 Measurement of the affinities for the CD160
protein, of the anti- CD160 H7 antibody and of the variants thereof
in the IgG1 format Reference IgG, No. KD (M) kon(1/Ms) kdis(1/s)
RMax Full X{circumflex over ( )}2 Full R{circumflex over ( )}2
ELB02101 H7 2.94E-09 3.70E+05 1.09E-03 0.533 0.1222 0.9923 IgG1
ELB02102 1.55E-09 4.22E+05 6.54E-04 0.9513 0.2779 0.9958 ELB02103
1.43E-09 3.71E+05 5.31E-04 0.8735 0.237 0.9955 ELB02104 1.47E-09
4.42E+05 6.51E-04 0.8788 0.3971 0.9933
TABLE-US-00010 TABLE 10 Measurement of the affinities for the CD160
protein, of the anti-CD160 H7 antibody and of the variants thereof
in the IgG1 E345K format Reference IgG, No. KD (M) kon(1/Ms)
kdis(1/s) RMax Full X{circumflex over ( )}2 Full R{circumflex over
( )}2 ELB02101 H7 2.94E-09 3.70E+05 1.09E-03 0.533 0.1222 0.9923
IgG1 ELB02112 1.52E-09 4.47E+05 6.79E-04 1.2637 0.8515 0.993
ELB02113 1.55E-09 4.27E+05 6.62E-04 1.1864 0.6878 0.9934 ELB02114
1.93E-09 4.27E+05 8.25E-04 1.1987 0.6949 0.9931
Regardless of the variant, and regardless of the nature of the
isotype (IgG4, IgG4 H310A-H435Q, IgG1 and IgG1 E345K), the variants
always have at least a 2-fold improvement in affinity for the
recombinant CD160 compared with the corresponding H7, a biolayer
that is twice as thick reflecting a better k.sub.on and a
dissociation constant which is two times lower than the
corresponding parental H7.
The H7 variants, regardless of the variant and regardless of the
nature of the isotype (IgG4, IgG4 H310A-H435Q, IgG1 and IgG1 E345K)
have better kinetic characteristics than H7.4.3-Biacore (SPR)
measurement for comparison of the affinity for the recombinant
human CD160, of H7 and of the different variants thereof, this
being in different IgG formats
In order to compare the affinity for the recombinant human CD160,
of H7 and of the different variants thereof, Biacore (SPR)
measurements were also carried out as described in a design close
to that described in Example 2.
TABLE-US-00011 TABLE 11 Affinities of the recombinant human
CD160/anti-hCD160 interaction of the H7 antibody and the variants
thereof in different formats measured by Biacore (SPR) and by
Biolayer interferometry (BLI) Measure- Gain in Chi2 (SPR) ment kon
kdis Rmax KD KD/respective or Full R{circumflex over ( )}2
Anti-CD160 code method (1/Ms) (1/s) (RU) (nM) IgG Full X{circumflex
over ( )}2 (BLI) BLI ELB02101 H7 IgG1 WT SPR 2.09E+05 2.51E-03 630
12 12.60 BLI 3.70E+05 1.09E-03 0.533 2.94 0.12 0.99 ELB02101 H7
IgG1 D12 SPR 2.51E+05 1.13E-03 1200 4.5 2.7 57.70 BLI 4.22E+05
6.54E-04 0.9513 1.55 1.9 0.28 1.00 ELB02101 H7 IgG1 F04 SPR
2.00E+05 7.26E-04 1280 3.6 3.3 23.30 BLI 3.71E+05 5.31E-04 0.8735
1.43 2.1 0.24 1.00 ELB02101 H7 IgG1 A09 SPR 2.23E+05 8.77E-04 1250
3.9 3.0 36.30 BLI 4.42E+05 6.51E-04 0.8788 1.47 2.0 0.40 0.99
ELB01101 H7 IgG4 WT SPR 2.18E+05 2.85E-03 533 13.1 15.40 BLI
4.69E+05 1.23E-03 0.0021 2.61 0.11 0.99 ELB01103 H7 IgG4 D12 SPR
2.92E+05 1.22E-03 990 4.2 3.1 54.40 BLI 4.94E+05 7.52E-04 0.0026
1.52 1.7 0.27 0.99 ELB01102 H7 IgG4 WT SPR 2.05E+05 2.97E-03 636
14.5 11.50 S228P/R409K/ BLI 5.07E+05 1.48E-03 0.0019 2.91 0.11 0.99
H310A/H435Q ELB01104 H7 IgG4 D12 SPR 2.44E+05 1.23E-03 1020 5 2.9
34.40 S228P/R409K/ BLI 4.99E+05 7.44E-04 0.0028 1.49 2.0 0.31 0.99
H310A/H435Q ELB01106 H7 IgG4 F04 SPR 1.76E+05 7.21E-04 1010 4.1 3.5
8.55 S228P/R409K/ BLI 4.53E+05 7.36E-04 0.0024 1.63 1.8 0.18 1.00
H310A/H435Q
In Table 11, it is seen that the results obtained for
anti-CD160/CD160 interaction measurement with a second technique
(SPR) demonstrate, just like the measurements of affinity of these
same antibodies by BLI, that the gain obtained for the H7 variants,
regardless of the nature of the isotype (IgG4, IgG4 H310A-H435Q,
IgG1 and IgG1 E345K), is always at least a 2-fold improvement in
affinity for the recombinant CD160 compared with the corresponding
H7, an Rmax that is two times higher, reflecting a better k.sub.on,
and a dissociation constant that is two times lower than the
corresponding parental H7.
EXAMPLE 5
Binding of the Anti-hCD160 H7 and of the H7 Variants Thereof in the
IgG4 and IgG1 Format and in the IgG1 E345K Format on CHO CD160
Cells and on Non-Transfected CHO Cells and on NK Cell Line YT2C2
CD160 Cells
The binding capacity of the anti-CD160 H7 antibodies and of the
variants (D12 and A09) thereof in the IgG4 and IgG1 format and in
the IgG1 E345K format was evaluated during the labelling of surface
CD160 expressed in a recombinant line CHO-S-hCD160 (clone 2G10) in
comparison with non-transfected CHO-S cells, by measuring the
median fluorescence index (MFI) (see FIG. 1). For this,
5.times.10.sup.+5 2G10 (CHO-S-CD160) and non-transfected CHO-S
cells were labelled with 2 .mu.g of each of these antibodies and
also with the appropriate control isotypes. In FIG. 1, all the
anti-CD160 tested (regardless of the isotype or the IgG format or
the variant) specifically recognize human CD160 expressed
recombinantly by the CHO-S cells.
In FIG. 1A, IgG1 variants bind more efficiently to the CHO-hCD160
transfectants than the H7 IgG1 (which results in a median
fluorescence increased by a factor of 3 compared with H7 IgG1).
This is true for the IgG1 or IgG E345K format. The presence of the
E345K mutation in the Fc of the variants does not improve their
binding to these cells.
In FIG. 1B, the IgG4 variants bind more efficiently to the
CHO-hCD160 transfectants than H7 (which results in a median
fluorescence increased by a factor of 2 compared with H7 IgG4
ELB01101 or with H7 IgG4 H310A-H435Q ELB01102), this being even in
the presence of the H310A-H435Q mutations. Indeed, the presence of
the H310A-H435Q mutations in the Fcs of the anti-CD160s does not
impede their binding to their target, as can be seen when the
binding is compared between ELB01101 and ELB01102.
The binding capacity of the anti-CD160 H7 antibody and of the H7
D12 variant in the IgG4, Fab and Fab-linker Fab format was
evaluated during the labelling of surface CD160 expressed naturally
on a clone of NK cell line (YT2C2), by measuring the percentage of
cell labelled=percentage of binding (see FIG. 1C). For this,
2.times.10.sup.+5 YT2C2 cells were labelled with increasing
concentration of antibodies (from 50 nM to 0.39 nM) and also with
the appropriate control isotype. These results were analyzed using
the GraphPad Prism software for generating non-linear regression
curves (Log(agonist) vs response, 3-parameter equations) and
calculating the median effective concentrations (EC.sub.50). In
FIG. 1C, IgG4 variants ELB01103 and ELB01104 bind more efficiently
to the YT2C2 cells than the H7 IgG4 ELB01101 (which results in a
EC.sub.50 increased by a factor of 10 compared with H7 IgG4). This
is also true for the Fab-linker-Fab format ELB01132. By contrast
Fab format ELB01122 bind less efficiently to the YT2C2 cells than
the H7 IgG4 ELB01101 (which results in a EC.sub.50 decreased by a
factor of 2 compared with H7 IgG4). This is certainly due to the
monovalency of the Fab format.
EXAMPLE 6
Effect of the Anti-CD160 Antibodies According to the Invention on
the Inhibition of HUVEC Tube Formation and Characterization of the
Induction of CD160 Expression on HUVECs
Ten antibodies were evaluated for their effects on vascular tube
formation induced by VEGF or FGF in a Cell Player GFP-AngioKit
assay (Essen Biosciences). This set of samples comprises the
Avastin anti-VEGF antibody and the Lucentis antibody fragment.
Frozen human umbilical vein endothelial cells (HUVECs) pre-labelled
with a fluorescent protein (Green Fluorescent Protein or GFP) using
a lentiviral expression system were thawed and co-cultured with
human dermal fibroblasts on six 96-well assay plates for 2 days.
The antibodies and the reference agents (VEGF, FGF-2, control
medium) were added to various wells at various concentrations and
the assay plate was then placed in an IncuCyte live cell imaging
system. Fluorescent and phase-contrast (10.times.) images were
taken every 12 h for 10 days and analysed for the length of the
tube and the number of branching points. The culture media (with
antibody if required) and the assay supernatants were replaced
every 2-3 days.
EXAMPLE 7
Evaluation of the Efficacy of the Subconjunctival Injection of the
Anti-CD160 H7 Candidates in the IgG4 Format (ELB01101) in
Comparison with Aflibercept (Eylea.RTM.) in a Corneal
Neovascularization Model in the Rat
A corneal neovascularization model was developed in the rat. This
model in particular allows easy observation of the monitoring of
the appearance of neovessels in the cornea, which enables an
evaluation of molecules having anti-angiogenic properties,
including the antibodies according to the invention.
The antibody batches were generated, produced, purified and
qualified according to the process for preparing batches for
intravitreal and subconjunctival injections.
Groups of 6-week-old male Lewis rats were used.
Induction of Corneal Neovascularization in the Rat
D.sub.0: Surgical intervention under an operating microscope is
performed on one eye of each of the rats, after anaesthesia. For
this, the cornea is entirely de-epithelialized by application of
ethanol at 70.degree. C., followed by an incision of the limbus,
which leads to the appearance of corneal neovessels from D4.
Only the right eye is used. The animals are anaesthetized with an
injection in the right femoral muscle of ketamine (Imalgene 500),
100 .mu.l per rat and xylazine (Rompun 2%), 100 .mu.l per rat. A
drop of tetracaine is instilled in the right eye. The manipulations
are carried out under an operating microscope. The
neovascularization is induced by destroying the epithelium of the
cornea by applying a "microsponge" soaked in 70% alcohol to the
surface of the cornea. In parallel, a thickness of approximately
1.5 mm of conjunctiva is removed around the limbus with
microscissors. An antibiotic ointment (Fucidine) is applied to the
eye. The pupils are then kept closed for 4 days after suturing (5-0
silk thread). After 4 days, the pupils are opened by removing the
threads; the change in the neovessels of the cornea is examined
under an operating microscope on D4, D8 and D12 after
anaesthesia.
Treatments
10 rats per group (except 3 for the IgG4 control isotype) are used
in the following way:
D.sub.0: The operation is performed on one eye of the animals, as
described above;
D.sub.8: Photos are taken and the animals are divided into 8 groups
of 10 rats each, in order to be treated on the eye in which the
operation was performed:
The animals are injected with the products subconjunctivally using
a syringe fitted with a 291/2 G needle (Myjector) on D4 and D8.
Group 1: subconjunctival injection of 50 .mu.l of PBS (negative
control), Group 2: subconjunctival injection of 250 .mu.g of
Aflibercept.RTM. (Eylea) in 50 .mu.l, Group 3: subconjunctival
injection of 500 .mu.l of IgG4 control isotype in 50 .mu.l, Group
4: subconjunctival injection of 500 .mu.g of H7 IgG4 antibody in 50
.mu.l according to the invention
On D8 and D12: Photos of the eyes on which the operation was
performed are taken after observation under an operating microscope
in order to evaluate the effect of the treatment on the
neovascularization of the cornea. Samples of the sera and the
vitreous humours are taken on D+12 post-mortem.
The rat eye photos (.JPEG) were analysed using software (Calopix,
TRIBVN). The analysis was carried out blind, without knowledge of
the group or of the timing of the photograph. The evaluation of the
vascularization is determined using quantification software. The
vascularization was estimated as being the surface area of the
blood vessels relative to the total surface area of the eye
analysed (that is to say the de-epithelialized area). The photos
are reported and the % neovascularizations relative to the total
surface area are reported in the graph of FIG. 2.
Results:
Photographs of the eye on which the operation was performed are
taken on D0 and on 2 different days: D7 and D12. The photos taken
show the change in the corneal neovascularization, in particular
the development of the vascular density and of the length of the
vessels up to D12 in the isotype control.
The results presented in FIG. 2 show a decrease in the vascular
density in the animals treated with the H7 monoclonal antibody
according to the invention, compared with the animals injected with
a negative control, in this case the IgG4 control isotype.
It is also seen that the dose of H7 IgG4 (ELB01101), despite a weak
cross-reactivity for rat CD160 (data not shown), reduces the
corneal neovascularization in this rat model in a manner comparable
to a dose of 250 .mu.g of a high-affinity soluble receptor for
VEGF, the fusion protein (Aflibercept.RTM.), an anti-angiogenic
used for the treatment of age-related macular degeneration (ARMD).
This was also obtained with the H7 antibody in the IgG1 N297Q
format.
The H7 antibody, in the IgG4 and IgG1 N297Q formats, according to
the invention thus has anti-angiogenic activity.
EXAMPLE 8
Comparison of the Systemic and Ocular Pharmacokinetic (PK) Profiles
of Various Formats of Anti-hCD160 ELB's Candidates in Rabbits After
Intravitreal and Intravenous Administrations with the Ones of
Parental CL1-R2, of Bevacizumab and of Ranibizumab
The objective of this study was to compare the systemic and ocular
pharmacokinetic (PK) profiles of various formats of anti-hCD160
ELB's candidates (including the ELB011 candidates) with those of
the parental murine IgG1 anti CD160 CL1-R2 and of bevacizumab. To
screen for an optimized anti CD160 candidate that will have a good
time of residence in the eye and the lowest systemic half-life, a
PK study using 54 pigmented rabbits (HY79b strain) was performed.
The same dose (0.5 mg) of item to be tested was administrated via
intravitreal (IVT) or intravenous (IV) injections in rabbits, and
seric antibody concentrations were determined by LC-MS/MS for
ELB011 leads (and for ELB02104 and ELB02114 anti CD160 mAbs from
the ELB021 program) or by using commercial ELISAs for CL1-R2 and
for IgG and Fab comparators, here respectively the bevacizumab
(Avastin) and the ranibizumab (Lucentis) in their marketed
formulations. This allowed to modelize pharmacokinetic parameters
after IV bolus of each candidate and thus to calculate output
kinetic parameters for each drug.
Method for Intravitreal Injection (Tested Groups and PK Blood
Samples)
The study of the pharmacokinetics (PK) of the various anti-CD160s
and of their controls after a single administration of 500 .mu.g by
intravitreal (IVT) injection is carried out in New Zealand white
rabbits
The study was carried out with rabbits in good health (bacterial
and viral status of the animal known, one sex) (KBL Charles River)
(2750-3000 g of body weight, age at the beginning of treatment:
14-18 weeks). These animals were placed in a cage in the
conventional care unit for one week before randomization and for 4
consecutive weeks during the study (the animals will be housed at 1
to 2 animals/cage). The experimental protocol was subjected to the
ethics committee of the provider before beginning.
The experimental design of the experiment is described in Table 12.
This experiment contains 8 groups of 3 rabbits/group including 6
different formats of anti-CD160 (see Table 12 below) at a
concentration of 5 mg/ml with an endotoxin level of 0.5 EU/ml and 2
controls, bevacizumab (Avastin) and ranibizumab (Lucentis).
The administrations of drugs (50 .mu.l/eye/administration) were
performed by a bilateral injection of 250 .mu.g of each drug in a
final volume of 50 .mu.l under general anaesthetic.
The rabbits were followed by a weekly monitoring of the clinical
signs and of the body weight and by Ocular observations for ocular
tolerance in order to search for macroscopic signs of ocular
irritation (even minimal) and extensive ophthalmological
examinations of the back of the eye (using slit lamp and indirect
ophtalmoscope for the integrity of the characteristics of the head
of the optic nerve, (retinal and choroidal) vascular network and
RPE and Bruch's pigmentation membranes/coloration.
After the death of the animals, both eyes are enucleated and
immediately frozen at -80.degree. C. Before analysis, the frozen
eyes are separated into three parts--the vitreous body, the aqueous
humour, and the retina/choroid. The volume of the aqueous humour
samples and of the vitreous samples (after homogenization and
centrifugation) is measured. The frozen retina/choroid were
weighed.
Approximately 0.5 ml of total blood samples from the central artery
of the ear of the rabbits were taken in a tube without
anticoagulant before administration (pre-dose T0) and after
administration at 2 h, 6 h, 12 h, 24 h, 48 h (D2), 96 h (D4), 168 h
(D7) and 336 h (D14). The serum was stored frozen until analysis.
The serum samples were analysed to determine the anti-CD160
concentration.
Method for Intravenous Injection (Groups Tested and PK Blood
Samples)
The experimental design of the experiment is described in Table 12.
This experiment contains 10 groups of 3 rabbits/group including 8
different formats of anti-CD160 (see Table 12 below) at a
concentration of 5 mg/ml with an endotoxin level of 0.5 EU/ml and 2
controls, bevacizumab (Avastin) and ranibizumab (Lucentis).
The administrations of drugs were performed by a single bolus
intravenous injection of 500 .mu.g in maximum 50-200 .mu.l under
general anaesthetic (for whole IgG or molar equivalent for the mAb
fragment).
Approximately 0.5 ml of total blood samples were collected in a
tube without anticoagulant before administration (pre-T0) and after
administration at 5 min, 15 min, 30 min, 60 min, 2 h, 6 h, 12 h, 24
h, 48 h (D2), 96 h (D4), 168 h (D7) and 336 h (D14). The serum is
stored frozen until analysis. The serum samples were analysed to
determine the anti-CD160 concentration.
TABLE-US-00012 TABLE 12 Groups of the pharmacokinetic study in
rabbits after an IV and IVT administration of 0.5 mg (0.19 mg/Kg)
Injection Injection route and Number of route and Number of
Treatment group frequency animals frequency animals 1 ELB01101 (H7
G4) 250 .mu.g/ 3 500 .mu.g/ 3 2 ELB01103 (D12 50 .mu.l IVT, 3 100
.mu.l 3 G4) bilateral bolus 3 ELB01104 (D12 once on 3 on D0 3 H310A
- R435Q) D0, 4 ELB01122 (D12 3 3 Fab) 5 ELB01132 (D12 3 3 Fab
linker Fab) 6 CL1-R2 3 3 7 ELB02104 (D12 none 3 IgG1) 8 ELB02114
(D12 none 3 IgG1 E345K) 9 Bevacizumab 3 3 10 Ranibizumab 3 3
Bioanalysis of the Concentration of Each of the ELB011 and ELB021
H7-Derived Anti CD160 mAbs and Fragments Over Time in Rabbit Sera
Samples
The quantification of the different ELB011 anti human CD160
candidates (as intact IgG and IgG fragments) in rabbit serum
samples following two routes of injection (intravenous (IV) vs
intravitreal (IVT)), was performed using a high-performance liquid
chromatography-tandem mass spectrometry (LC-MS/MS) method. The
method development strategy aimed at obtaining one generic LC-MS/MS
method suitable for the determination of the concentration of all
ELB011 (and ELB021) drugs in rabbit serum. The samples were
prepared by enrichment in drug of interest by Protein L affinity
purification on magnetic beads, followed by reduction and
alkylation using DTT and iodoacetamide, prior trypsin digestion.
The final extract was analysed via HPLC with MS/MS detection using
positive ion electrospray. One tryptic peptide
(ASQSISNHLHWYQQKPGQAPR residues 25-45 of SEQ ID NO: 14 including
one CDR of the VL light chain) common to all anti CD160 (from
ELB011 and ELB021 programs) based on H7 humanized candidate was
monitored in the multiple reaction monitoring (MRM) method. The
chosen peptide maps in the CDR region of the light chain, allowing
the direct transfer of the assay to other pre-clinical matrices and
possibly to analysis in human matrices. Then, the method was first
qualified and then applied to the quantification of each compound
in the rabbit sera.
Two other anti CD160 mAbs (ELB02104 and ELB02114, H7-A09 anti CD160
as IgG1 and hexameric IgG1E345K format respectively) for which only
IV route on injection was tested were also quantified according to
the same method.
Bioanalysis of the Concentration of CL1-R2, Bevacizumab and of
Ranibizumab Over Time in Rabbit Sera Samples
To compare pharmacokinetic parameters of H7 and variants of H7 to
those of parental murine anti human CD160 CL1-R2, mouse IgG1
concentrations in rabbit sera were measured using enzyme linked
immunosorbent assay (ELISA) using the commercial Mouse IgG1 ELISA
Quantitation Set (Cat. No. E90-105, Lot No. E90-105-39 from Bethyl
Laboratories) according to manufacturer's recommendations.
Ranibizumab and bevacizumab were chosen as comparators due to their
molecular format (IgG1 and Fab respective). These compounds were
dosed in rabbit sera using respectively Biovision's E4312-100 for
ranibizumab (Lucentis)'s dosing and Biovision's K4254-100 for
bevacizumab (AVASTIN)'s dosing.
Methods Used for Pharmacokinetic Parameters Analysis
For both injection routes, using a non-compartmental analysis, the
following pharmacokinetic parameters were observed and calculated
using measured seric concentrations:
For all serum concentrations time curves C Max (.mu.g/ml) (serum
peak concentration occurring in a profile), TMax (H) (Time of serum
peak concentration), TLag (H) (delay between drug administration
and first observed serum concentration (when sufficient data)),
AUC0-t obs (H..mu.g/ml) (Area under the serum concentrations time
curve from 0 to Clast, using the log-trapezoidal rule), AUC0-inf
obs (H..mu.g/ml) (Area under the serum concentrations time curve
extrapolated from 0 to infinity (AUC0-t+Clast/Ke)), Clast (last
observed conc), Elimination Ke (H-1) (slope of the terminal part of
the serum concentration times-curve, obtained by log-linear
regression (if sufficient data)), Terminal T.sub.1/2 (H) (observed
elimination half-life or terminal half-life, calculated as
"T.sub.1/2=-In 2/Ke"), Vd (Distribution volume (L) after IV Bolus
Vd=dose/(Ke.times.AUC0-inf) (IV Bolus only)), CL (Clearance (L/H)
CL=Ke.times.Vd (IV Bolus only)).
For items that were injected by IVT only, additional parameters
were evaluated like F % (AUC0-t) (Absolute bioavailability to the
reference IV Bolus=AUC0-t Test.times.Ref dose/AUC0-t Ref.times.Test
dose) and F % (AUC0-inf) (Absolute bioavailability to the reference
form=AUC0-inf Test.times.Ref dose/AUC0-inf Ref.times.Test dose (If
AUC0-inf measurable)).
Then, an IV bolus pharmacokinetic two-steps modelization was
performed to calculate the parameters and rate constants of the
kinetics in order to provide a compartmental model and information
on the intrinsic disposition of the drug (distribution and
elimination) (as described in Wagner, J. G. 1975). The disposition
model of each candidate and the model dependent pharmacokinetic
parameters of IV BOLUS doses are useful for the calculation of the
IVT output after an IVT dose by deconvolution methods. This
modelization was performed to determine the pharmacokinetic model
in serum in each rabbit for each tested item.
At this stage, the kinetic of ocular elimination of drugs in the
serum and the fraction of the dose eliminated in serum after IVT
administration are calculated by a compartmental deconvolution
method using the calculated IV bolus disposition model of the drug.
If 2 or more compartments are observed, the Loo-Riegelman method
(Loo J C, Riegelman S., 1968) is applied, if 1 compartment is
observed, the Wagner Nelson (Wagner J G, Nelson E. 1968) is
applied. The results are the cumulative quantity of drug which
enter the serum (drug input) and the rate (drug input rate) versus
the time. After an IVT administration of drugs the cumulative serum
input profile is the ocular output profile and the rate is the
output rate. In the meantime, the other following parameters could
be also determined. Tlag (H) The Lag Time (when sufficient data)
Ocular Output (mg): the total unchanged quantity of drug released
in the serum Output (% Dose): The % Dose of drug released in the
serum (this is the absolute bioavailability of the ocular dose
after IVT) Output Time of 50% (.+-.): Time to observe 50% of the
injected dose which have entered the serum (graphic estimation)
Time of % Max (H): Time to reach the plateau of the cumulated drug
output kinetic=the duration time of ocular residence of the drug
Output Rate (mg/H): rate of drug input in the serum Max Output Rate
(mg/H): Peak of the Output rate curve Time of Max Output Rate
(H):Time of peak.
TABLE-US-00013 TABLE 13 Main observed pharmacokinetic parameters
after a 0.5 mg (0.19 mg/Kg) dose administered intravenously (IV)
Cmax mean AUC TESTED Molecular T.sub.1/2 Serum. Cmax. ratio/ Tmax.
AUC0-t ratio/ ITEM structure day .mu.g/mL ELB01101 Hour H micrg/ml
ELB01101 ELB01101 Anti CD160 IgG4 7.84 4.05 1.00 0.14 248.12 1.00
ELB01103 Affinity matured 2.68 3.67 0.91 0.44 163.64 0.66 IgG4
ELB01104 Affinity matured 2.17 3.66 0.90 0.19 113.94 0.46 IgG4 FcRn
null mutation ELB01132 Fab-linker Fab 0.19 3.46 0.85 0.08 13.28
0.05 ELB01122 Fab 0.08 2.26 0.56 0.08 3.18 0.01 ELB02104 Anti CD160
IgG1 2.82 4.30 1.06 0.08 146.11 0.59 ELB02114 Affinity matured 1.67
3.36 0.83 0.08 95.74 0.39 IgG1 E345K Bevacizumab anti VEGF human
3.08 6.91 1.71 0.39 464.60 1.87 IgG1 CL1-R2 anti CD160 IgG1 2.27
6.98 1.72 24.00 541.39 2.18 murin
TABLE-US-00014 TABLE 14 Main observed pharmacokinetic parameters in
sera after a 0.5 mg (0.19 mg/Kg) dose administered intravitreally
(IVT) Cmax mean AUC TESTED Molecular Cmax, ratio/ Tmax, AUC0-t
ratio/ ITEM structure .mu.g/mL ELB01101 days H micrg/ml ELB01101
ELB01101 Anti CD160 IgG4 0.79 1.00 7.00 96.05 1.00 ELB01103
Affinity matured 0.67 0.85 3.33 83.91 0.87 IgG4 ELB01104 Affinity
matured 0.64 0.81 4.33 54.84 0.57 IgG4 FcRn null mutation ELB01132
Fab-linker Fab 0.11 0.14 2.00 9.87 0.10 ELB01122 Fab 0.04 0.05 1.33
3.45 0.04 Bevacizumab human IgG1 1.74 2.21 168.00 303.26 3.16
CL1-R2 murine IgG1 0.57 0.72 104.00 70.76 0.74
As shown in tables 13 (IV route) and 14 (IVT route), the expected
seric PK profiles (i.e. the main observed seric pharmacokinetic
parameters) of ELB011's and ELB021's candidates after a 0.5 mg
(0.19 mg/Kg) dose administered intravenously (IV) or intravitreally
(IVT) and the ranking of their seric half-life (also noted
T.sub.1/2) are in accordance with the expected differences due to
their corresponding formats (as described previously by Gadkar et
al., 2015).
IV Bolus Observations and Modelization
As seen in FIG. 3A, after an IV bolus, the serum concentrations of
ELB01101, ELB01103, ELB01104, ELB01122, ELB01132, ELB02104 and
ELB02114 decrease rapidly post injection (distribution phase),
followed by a 2nd step of classical slow elimination phase
(T.sub.1/2 range: 52 to 188 H) excepted for ELB01122 and ELB01132
(1.9 and 4.6 H). As shown in table 13, the CMax (3,360 to 4,297
.mu.g/ml) are observed at the first sampling time 0.083 H (2
minutes) for ELB01122, ELB01132, ELB02104 and ELB02114 but after a
longest time for ELB01101, ELB01103 and ELB01104 (from 0.139 to
0.444 H). The mean distribution volume and clearance vary
respectively from 0.05 to 0.33L and 0.005 to 0.1051 L/H.
As expected, anti CD160 fragments have reduced seric PK parameters
after IV injection compared to IgG like formats. In particular,
ELB01132's seric half-life seems to be slightly higher than the
seric half-life of ELB01122.
Regarding pharmacokinetic parameters after IV administration of
bevacizumab, when observed data are compared to literature data,
the observed seric T.sub.1/2 of bevacizumab in rabbit serum is
quiet low compared to literature (3 vs 5.32 days, EMEA file for
bevacizumab (AVASTIN)).
Regarding the ELB011 and ELB021 candidates in IgG formats, ELB01101
has the longest seric half-life, even longer than the one of CL1-R2
or bevacizumab, (Avastin). ELB01103 and ELB02104 have roughly the
same seric half-life than bevacizumab, while ELB01104, ELB02114 and
CL1-R2 have a reduced seric half-life compared to bevacizumab and
ELB01103 and ELB02104. But, when AuC, Cmax and Tmax parameters are
compared with those of bevacizumab and of CL1-R2, these parameters
are significantly reduced with all the ELB011 candidates (and also
with ELB021 candidates). Indeed, the serum concentrations of CL1-R2
and bevacizumab are not similar to the ones ELB011's & ELB021's
candidates, CMax are highest: 6,980 and 6,091 .mu.g/ml at 0.389 H
for CL1-R2 and 24.7 H for bevacizumab (unexpected TMax
corresponding to a slow infusion of bevacizumab). T.sub.1/2, Vd and
CL are respectively for CL1-R2 and bevacizumab: 54.5 and 74 H; 0.05
and 0.09 L; 0.0010 and 0.0009 L/H. This could due to the fact that
bevacizumab and CL1-R2 have probably a lowest catabolism.
When pharmacokinetic parameters of ELB01101 (the non-affinity
matured variant) are compared to those of the other affinity
matured ELB011's and ELB021's candidates, the affinity maturation
of anti CD160 mAb has decreased the serum half-life of these
antibodies in rabbits. Indeed, the clearance in serum of all the
affinity matured candidates is increased, this is shown in
particular for ELB01103, ELB01104, ELB02104 and ELB02114 for which
half-life in sera is significantly reduced compared to the one of
ELB01101, and of bevacizumab. This faster clearance could be due to
a CD160 driven specific biological process like for example an
increase of anti CD160 mAb internalisation on CD160 positive cells
in the blood. This should be checked in other species to be certain
of the impact on clearance of anti CD160 due a high affinity for
CD160.
ELB01103 and ELB02104 have a seric T.sub.1/2 equivalent to the one
of ELB01104 (the affinity matured variant with additional FcRn null
mutations to prevent IgG recycling via neo natal FcRn receptor)
rather than the one of the non-affinity matured native ELB01101. In
the case of ELB01104, (as expected and previously described in
(Olafsen, 2012) the FcRn mutations seem to have slightly reduced
the seric half-life of ELB01104 compared to the one of ELB01103.
But, as pharmacokinetic parameters of one ELB01101 with the same
FcRn null mutations are not available, it is difficult to evaluate
the impact of FcRn mutations alone. However, the PK profile
differences between ELB01103 and ELB01104 versus ELB01101 are more
important than the one between ELB01103 and ELB01104. The
consequences of affinity maturation on ELB01104's PK parameters
have more impact than the consequences of a reduced IgG
recycling.
IVT Observations
As described in FIG. 3B, the serum concentrations increase slowly
post injection in accordance to a slow output of the drugs from the
eye, the highest profile level is observed after intravitreal
administration of bevacizumab and the lowest after intravitreal
administration of ELB01122 and ELB01132.
As reported in table 14, the mean CMax are in a range of 0.567 to
0.787 .mu.g/ml for CL1-R2, ELB01103, ELB01104 and ELB01101 but
lowest for ELB01122 and ELB01132 (0.041 and 0.114 .mu.g/ml) and
highest for bevacizumab (1.736 .mu.g/ml). The TMax are observed
between 48 and 168 H, the T.sub.1/2 are indeterminable in all
rabbits (no or insufficient data after the end of the ocular output
to observe the serum elimination slope).
When administered intravitreally, all the ELB011 candidates, as
well as the parental CL1-R2, have a lower systemic Cmax, a reduced
Tmax and a reduced mean AuC compared to bevacizumab. As expected,
the ELB011 anti CD160 fragments (ELB01132 and ELB01122) have
reduced observed seric PK parameters compared to all the IgG
formats injected intravitreally. These fragments are eliminated
from the bloodstream more rapidly than all the IgGs based molecules
(seric T.sub.1/2 reduced at least by 30 fold compared to ELB01101).
After an IVT injection, ELB01132's seric half-life seems again to
be slightly higher than the seric half-life of ELB01122, as seen
after IV injection.
After IVT injection, the ELB011 IgG candidates (ELB01101, ELB01103
ELB01104) have observed seric PK parameters close to the ones of
CL1-R2 and are the ones expected for an intravitreally administered
IgG format (as described in (Gadkar et al., 2015), for example).
Tmax are the ones expected for each format, excepted for ELB01103.
ELB01103 has an intermediate behavior (as shown by its PK
parameters) between an IgG4 (ELB01101) and an IgG with no recycling
via FcRn (ELB01104). When compared to literature, here again as
observed after IV injection, data from Gadkar et al 2015, impact of
FcRn mutations on Cmax, Cmax ratio.sub.(Versus ELB01101), Seric AuC
o-t and Seric AUC ratio.sub.(Versus ELB01101) of ELB01104 is less
pronounced than expected.
IVT Output Kinetics Determination
TABLE-US-00015 TABLE 15 Pharmacokinetic parameters in rabbits of
ELB011 candidates after a deconvolution analysis of mean sera
concentrations following both routes of administration of each item
Pharmacokinetic parameters in rabbit after a 0.5 mg IVT (~0.19
mg/Kg) administration Time of % Max mean max % (Day) = serum Tmax
AUC0-t of initial OCULAR OUTPUT Output time of ELB0111 Cmax, serum,
H IVT dose in OUTPUT (% Time of residence candidates .mu.g/mL days
micrg/ml sera * (mg) Dose) 50% Day in eye ELB01101 0.787 7.00 96.05
19.67 0.341 68.150 5.000 7.000 ELB01103 0.670 3.33 83.91 16.75
0.258 51.510 3.000 4.000 ELB01104 0.637 4.33 54.84 15.92 0.346
69.260 2.667 3.667 ELB01132 0.114 2.00 9.87 2.85 0.201 40.280 ND
2.000 ELB01122 0.040 1.33 3.20 1.00 0.123 24.633 ND 1.667
Bevacizumab 1.736 7.000 303.260 43.400 0.420 83.970 5.000 7.000
CL1-R2 0.565 4.333 70.760 14.125 0.176 35.150 7.000 4.333 after a
0.5 mg IV after a 0.5 mg IVT BOLUS (~0.19 mg/Kg (~0.19 mg/Kg)
administration administration Max Time of mean Time OUTPUT Max
AUC0-t T1/2 residence ELB0111 Rate Rate H Day, In vitreous **/
candidates (mg/H) (Day) micrg/ml sera T1/2 sera ELB01101 0.004
2.000 246.123 7.837 0.893 ELB01103 0.006 2.000 163.640 2.678 1.494
ELB01104 0.009 1.667 113.943 2.173 1.687 ELB01132 0.006 1.333
13.280 0.192 10.412 ELB01122 0.004 1.333 3.177 0.081 20.513
Bevacizumab 0.004 2.000 464.600 3.082 2.271 CL1-R2 0.003 0.667
541.387 2.272 1.908 * Assuming a mean rabbit blood volume of 125
mLs at Cmax and Tmax ** based on time of % max
Based on the Cmax in serum after IVT injection and on an assumed
mean rabbit blood volume of 125 mLs for rabbits, it is also
possible to calculate the maximal concentration of product at Tmax
and expressed it as the maximum percentage of initial IVT dose in
sera at Cmax and at Tmax (Max % of initial IVT dose). This was
calculated for each of the anti CD160 candidates that were injected
by both routes and also for CL1-R2 and bevacizumab.
Regarding the calculated pharmacokinetic parameters summarized in
table 15, after IVT injections of 0.5 mg, the ocular output
corresponding to the calculated serum input of ELB01101, ELB01103,
ELB01104, CL1-R2 and bevacizumab are respectively 0.341, 0.184,
0.346, 0.176 and 0.420 mg corresponding to 68, 55, 69, 35 and 84%
of the vitreous dose respectively. The mean duration times of the
ocular output (Times of % max) are respectively 168, 96, 88, 104
and 168 H.
First, when ELB011 IgG (and fragments as well) candidates are
compared with bevacizumab, the systemic exposure is significantly
lower for all the ELB011 candidates (max 19% with ELB01101 and
16.75% for ELB01103 for example) than for bevacizumab (43%). It
means that, with ELB011 candidates, there is less amount of IVT
injected product that went in the systemic than with bevacizumab.
Indeed, the ocular output, the output dose and the maximum
percentage of the initial IVT dose are very high for bevacizumab
compared to all the ELB011 candidates.
Secondly, when ELB011's IgG candidates are compared to CL1-R2,
ELB01101 and ELB01104 have a slightly higher total ocular output
and output % of initial dose compared to ELB01103 and CL1-R2.
Neither FcRn null mutations nor affinity maturation seems to have a
major impact on ocular output and output dose. For ELB01103, there
is less amount of product (less than 50% of the initial IVT total
dose) that went into the serum compared to ELB01101 & ELB01104
(68 and 69%). ELB01103 is the IgG candidate that has a lower seric
half-life than ELB01101 so even if some product goes into the sera,
ELB01103 systemic product will be cleared 2 times faster than the
ELB01101 systemic product.
In terms of output rate, the output rates ranged from 0.004 to
0.009 mg/h. ELB01104 has the highest output rate then ELB01103 and
ELB01132 have the same output rate and finally ELB01101 and
ELB01122 have the lowest output rate.
When ELB011 fragments candidates are compared to the other ELB011
IgG candidates, it is clear that, globally, elimination of ELB01132
and ELB01122, after an intravitreal administration, is clearly
different from the one of anti CD160 in the IgG format. Both
fragments have a similar PK profile of elimination from the eye
(same total ocular output and almost the same output dose).
Furthermore, as ELB01132 and ELB01122 are eliminated more rapidly
than all the IgGs based molecules from the bloodstream, a very low
content of any of these two products is found in the systemic
compartment (less than a max % of the initial IVT dose of 2.85% at
the Tmax). This is also the case for Lucentis for which no product
was detected in our hand in any of the rabbit sera after an IVT
injection of 500 .mu.g in the same condition.
When ELB01132 and ELB01122 are compared together, ELB01132 is
eliminated differently than ELB01122. In terms of elimination after
IVT, ELB01122 is the fastest product to be eliminated from the
bloodstream. Indeed, it has the lowest % of initial IVT dose that
goes into the bloodstream and the lowest ocular output. There is a
little bit more ELB01132 product that is eliminated from the eye,
(the ocular output is two-fold the one of ELB01122). ELB01132 seems
to go into the bloodstream a little bit more rapidly than ELB01122.
ELB01132 has a longer T.sub.1/2 of elimination (seric half-life)
and this impacts the other PK parameters. 75% of the initial IVT
injected dose seems to stay in the eye for ELB01122 vs 60% for
ELB01132. However, it seems that the mean residence time in the eye
of ELB01132 is a little bit better than the one of ELB01122. So,
both fragments candidates offer a very favorable ratio of T.sub.1/2
in the eye on T.sub.1/2 in systemic, better than the one for IgG
candidates, but they have a lower time of residence in the eye than
the candidates in the IgG format.
Note: The drugs serum inputs from the deconvolution of ELB01132 and
ELB01122 are less accurate than for the other ELB011 IgG candidates
due to a lower number of timepoints with a detectable seric
concentrations, due to a very short seric half-life, and due to an
initial delay of product release from the eye.
Summary--Conclusion on All PK Parameters After IV & IVT
Administrations for ELB011 and ELB021 Candidates
The expected seric PK profiles (i.e. the main observed seric
pharmacokinetic parameters) of ELB011's and ELB021's candidates
after a 0.5 mg (0.19 mg/Kg) dose administered intravenously (IV) or
intravitreally (IVT) and the ranking of their seric half-life (also
noted T.sub.1/2) are in accordance with the expected differences
due to their corresponding formats (as described previously by
(Gadkar et al., 2015).
There is no impact of the FcRn null mutations or of affinity
maturation on the PK parameters in the eye.
ELB011 and ELB021 candidates have different seric PK parameters
than CL1-R2 and bevacizumab with a faster clearance in the serum,
but this should be checked in other species (mice and non human
primates in dedicated studies).
Regarding seric T.sub.1/2, the ranking is very similar to the one
of time of residence in the eye (as described in Gadkar & al,
2015),
ELB01101>>ELB01103.about.ELB01104>ELB01132.about.ELB01122.
The surprise came from ELB01103. ELB01103 has a behavior closer to
the an IgG4 with no FcRn binding than an IgG4. With the different
anti CD160 formats, the % of intravitreally injected antibody that
ends up in the systemic as well as the sericT.sub.1/2, were
massively reduced and systemic exposure after IVT of ELB011
candidates is equivalent to the one of CL1-R2 and lower than the
one of bevacizumab.
The total ocular output and the output % of initial dose of
IgG-like anti-CD160 ELB01101 and ELB01104 are high compared to the
ones of ELB01103.
In terms of % of the dose that stayed in the eyes, ELB01122 is
better than >ELB01132>ELB01103>ELB01104>ELB01101. In
terms of output rate from the eye, the output rates ranged from
0.004 to 0.009 mg/h. ELB01104 has the highest output rate then
ELB01103 and ELB01132 have the same output rate and finally
ELB01101 and ELB01122 have the lowest output rate.
In terms of time of residence in the eye (See column Time of % Max
in table 15) the ranking between ELB011's candidates is the
following one:
ELB01101>>ELB01103.about.ELB01104>>ELB01132.about.ELB01122.
Choice of the Leads to be Further Tested in In Vivo Preclinical
Model.
Among the potential ELB011 candidates, two leads were finally
chosen, one IgG fragment and one whole IgG to be compared in a dose
efficacy study in the adequate NHP model.
Regarding the screen for an optimized anti CD160 candidate that
will have a good time of residence and the lowest systemic
half-life, on the left hand, the ratio Time of residence in the
eye/T.sub.1/2 sera, for the IgG-like mAbs, is better for ELB01104,
then for ELB01103 and finally for ELB01101
(ELB01104>ELB01103>>ELB01101). But, on the other hand, for
ELB01104, 70% of the initial dose goes into the systemic (and only
50% for ELB01103), i.e. there is less product that stayed in the
eye for ELB01104 than for ELB01103. Thus, the final ranking between
ELB011 candidates as IgG format is
ELB01103>ELB01104>ELB01101.
For the Mab fragment choice, both fragments have a similar PK
profile of elimination from the eye (same total ocular output and
almost the same output dose). Both fragments candidates offer a
very favorable ratio of T.sub.1/2 in the eye on T.sub.1/2 in
systemic, better than the one for IgG candidates. It seems that the
mean residence time in the eye of ELB01132 is a little bit better
than the one of ELB01122. Finally, in terms of PK parameters and
taking into account other developability parameters (requirement of
avidity for an ideal CD160 binding and for questions of yield of
production and quality of produced fragments) the recommendation
was to select ELB01132 (Fab linker Fab) for further efficacy
testing.
EXAMPLE 9
Pilot Study of Efficacy/Tolerability of Intravitreal Injection of
the Anti-hCD160 H7 (IgG4 (ELB01101)) and H7 (IgG1 N297Q (ELB01111))
in a Model of Laser-Induced Choroidal Neovascularization in the
Non-Human Primate (NHP) Macaca fascicularis
The objectives of this study were (1) to determine the ocular
tolerance of two formats of anti-hCD160, H7 IgG4 (ELB01101) and
aglycosylated H7 IgG1 (ELB01111), when they are administered via a
single intravitreal injection in cynomolgus monkeys and (2) to
evaluate the potential preventive effect of one of these isoforms
on laser-induced choroidal neovascularization in a cynomolgus
(Macaca fascicularis) monkey model.
Justification of the Model Choice, Route of Exposure for Test
System and of the Number of Animals
Safety and dose assessment for efficacy (preventive effect) was
initiated in the most relevant NHP laser induced chNV model.
Indeed, this animal model has an established track record as a
predictor of pharmacologic efficacy of anti-neovascular drugs in
humans having the neovascular, or wet, form of age-related macular
degeneration.
The ocular route of exposure was selected because this is the
intended route of human exposure.
The cynomolgus monkey was chosen as animal model for this study
because it is a non-rodent species accepted for preclinical ocular
toxicity tests by the regulatory bodies. The total number of
animals to be used in this study is considered to be the minimum
required to correctly characterize the effects of the test
antibody. This study was designed such that it does not require a
needless number of animals in order to achieve its objectives.
The ocular tolerability, the clinical parameters and the preventive
effect of a single intravitreal injection of 1 mg per eye of the
ELB01101 IgG4 mAb, of ELB01111 (tolerability only) or of control
vehicle in laser-induced ChNV model were evaluated at Charles River
(Senneville, Canada). All the procedures used for this NHP model
study are the standard procedures from Charles River and are
briefly described below. Some minor changes were made compared to
the initial monkey model protocol initially developed by Ryan S J,
1982.
Study/Experiment Scheme
Animals and Animal Farming Conditions
A total of 17 male cynomolgus monkeys (from 2 to 3 years old) were
received and weighed between 2.7 and 3.2 kg at the initiation of
the dosage. A minimum acclimatization period of 4 weeks was allowed
between the reception of the animals and the beginning of the
treatment in order to accustom the animals to the laboratory
environment. The animals were housed socially (up to 3/group/cage)
in stainless steel cages equipped with an automatic watering valve.
Temperatures of 20.degree. C. to 26.degree. C. with a relative
humidity of 30% to 70% were normally maintained. A cycle of 12
hours of light/12 hours of darkness was maintained. The food was
provided in amounts appropriate to the size and age of the animals
(PMI Nutrition International Certified Primate Chow No. 5048 was
provided twice a day). The water after treatment by reverse osmosis
and ultraviolet radiation was freely available for each animal via
an automatic watering system. The monkeys were used in accordance
with the ARVO declaration for the use of animals in ophthalmic
research.
Experimental Design
As described in table 16, in a first phase, tolerability (global
and ocular tolerance) of intravitreal administration of 1 mg of the
H7 variant in two IgG formats (IgG4 and IgG1 N297Q) was compared in
3 non laser induced eyes of monkey.
In the second phase, the efficacy of the less toxic isoform or, if
equivalent tolerability, the H7 in its IgG4 format (ELB01101) in
the laser induced ChNV model was assessed.
TABLE-US-00016 TABLE 16 Summary of the experimental scheme phase 1
Tolerability and phase 2 Efficacy/extended tolerability Number Dose
Dose of Group Material tested Dose volume concentration animals No.
RE LE (mg/eye) (.mu.L/eye) (mg/mL) Males Phase 1 1 H7 H7 1.0 50 20
3 IgG4 IgG1 aglyc. Phase 2 2 PBS for injection 0 50 0 6 3 H7 IgG4
1.0 50 20 6 RE: right eye, LE: left eye.
Preparation of the Items to be Tested
On the day of use, the items to be tested (without aggregates and
with a very low endotoxin content (<0.025 EU/mg) (see Table 17))
were prepared at 20 mg/mL by dilution with the reference product
(PBS) at concentrations appropriate for meeting the dosage
requirements.
TABLE-US-00017 TABLE 17 Identification of the items and carrier
control tested Test item 1 Test item 2 (anti-CD160 (anti-CD160
Reference format 1) format 2) item/carrier Identification H7 IgG4
Aglycosylated PBS for H7 IgG1 injection Batch No. Prod 2 Prod * 28
Dec. 2015 4 Mar. 2016 Description Liquid Liquid Liquid Purity 99.5%
99.5% n/a Concentration 22.7 mg/mL 22.7 mg/mL n/a Endotoxin level
<0.5 EU/mL <0.5 EU/mL <0.5 EU/mL (Endosafe .RTM.-PTS .TM.;
<0.025 EU/mg <0.025 EU/mg Charles River) Storage conditions
2.degree. C. to 8.degree. C. 2.degree. C. to 8.degree. C. 2.degree.
C. to 8.degree. C.
Parameters Monitored
The following parameters were evaluated during this study:
mortality and clinical signs, body weight, change in body weight,
appetite, ophthalmology, fluorescein angiography, macroscopic
pathology and immunohistochemistry.
Procedures in Force, Observations and Measurements
The mortality/moribondity controls were normally carried out twice
a day, once in the morning and once in the afternoon, throughout
the study. Detailed examinations were carried out each week for the
dosage and observation periods. The individual body weights were
measured each week. The individual evaluation of the feeds was
evaluated daily by visual inspection of the general appetite.
Ophthalmological examinations were carried out during phase 1, once
in pre-study and on days 2, 5 and 7, and during phase 2, once in
pre-study after the induction of laser-induced CNV, on day 1, day 9
and again on day 28.
Ophthalmoscopic and biomicroscopic examinations (slit lamp). The
examinations were carried out by a certified
veterinarian-ophthalmologist. The mydriatic drops used were
tropicamide at 1%. A sedative, Ketamine.RTM. HCl for injection,
USP, was administered by intramuscular injection after an
appropriate fasting period.
Imaging Procedure
The development of active ChNV lesions was assessed by fluorescein
angiography (FA), once prestudy before injury and on days 14 and 29
after laser injury. The ChNV lesions defined by the individual
laser spots on the still images from days 14 and 29 were evaluated
for leakage semi-quantitatively.
Fluoro-Angiograms
During efficacy evaluation phase 2, imaging data (fluorescein
angiographies or fluoro-angiograms) were determined on day 1 (after
laser, in pre-dose) and were collected again on days 14 and 29
post-photocoagulation as follows:
Procedure: mydriatic drops (1% tropicamide) were applied to each
eye at least 25 minutes before the test. The hydration of the eyes
was maintained by frequent irrigation with a saline solution. The
animals receive an intramuscular injection of a sedative cocktail
of ketamine (5 mg/kg), glycopyrrolate (0.01 mg/kg) and
dexmedetomidine (0.01 mg/kg), and were then intubated with an
endotracheal tube in order to administer an isoflurane/oxygen
mixture. Once the angiography is finished, the animals receive, if
necessary, an intramuscular injection of 0.1 mg/kg of atipamezole,
a reversal agent for dexmedetomidine. The simple and/or real-time
retinal images in the free infrared and/or red modes were obtained
so as to act as reference images for the angiographies. 1.0 ml of
10% injectable fluorescein sodium U.S.P. was administered by rapid
intravenous injection (cephalic or saphenous vein), followed by a
water flush of 0.5 ml of saline solution. The fixed images were
recorded for the two eyes at least 2 minutes and at the latest 5
minutes after the fluorescein injection. In addition, the fixed
images of the two eyes were recorded at least 8 minutes and at the
latest 11 minutes after the fluorescein injection. In order to
ensure that the data are masked, the fluoro-angiography images were
identified by an animal arrival number, and not by the number of
randomized animals. The levels of severity of the lesions (grades
corresponding to each individual laser lesion) were evaluated on
the fixed images by the extent of the fluorescein leakage on a
scale of 0-4 by 2 masked and experienced independent readers, who
subsequently determine a consensus score using the following scale:
grade 0 no leakage, grade 1 minimal leakage, grade 2 slight
leakage, grade 3 moderate leakage (semisolid to solid
hyper-fluorescence generally remaining within the boundary of the
laser-induced defect region), grade 4 substantial leakage (solid
hyper-fluorescent region extending beyond the boundary of the
laser-induced defect region).
The total number and the % of relevant clinical lesions (grades 3
and 4) were counted totalled. Day 1 images were used for
confirmation of procedure and laser spot formation).
The number of clinically relevant lesions were defined by the
combination of the lesions of grade 3 and 4.
It is also possible to express the incidence on clinical relevant
lesions by the incidence rate and the incidence rate ratio as
defined in (Krzystolik et al., 2002). The incidence rate was
defined as the number of clinically relevant lesions (that occurred
during a given interval) divided by the total number of laser
induced lesions/spots. Incidence rate could be also expressed in
percentage. Then they calculated the incidence rate ratio (IRR)
that referred to the ratio of incidence rate of clinically relevant
lesions in the prevention eyes to the incidence rate in control
eyes. An IRR of 1 would signify no difference between incidence
rates. A IRR number much smaller than 1 would indicate a reduction
in the incidence of clinically relevant lesions in the prevention
group vs control group.
Immunohistochemistry by von Willebrand (vwf) Labelling
Following euthanasia, the eyes were enucleated and the vitreous
humor was collected and placed on dry ice followed by storage in a
freezer set to maintain -80.degree. C. Remaining tissue from the
left eyes from all phase 2 animals was used for
immunohistochemistry analysis. The choroid membranes of the
specified left eyes were prepared, mounted as "flat mount" and were
stained with von Willebrand factor (vWF) by a IHC study. Briefly,
flat mounts were washed in PBS+1% Triton buffer at least 5 minutes
3 times between each step, blocked in 1% BSA in PBS+1% Triton+0.1%
sodium azide for 30 minutes, submitted to a rabbit polyclonal to
von Willebrand Factor (1/200 of ab6994, Abcam) or to a negative
Reagent Control (1/350.times.0936 Dako/NRbIgG target for 48 h at
4.degree. C., and finally to a AlexaFluor 488 coupled goat
anti-rabbit IgG (A11008/Life Technologies) overnight at 4.degree.
C. The laser spot lesions treated or not by the anti-CD160 was
individually evaluated semi-quantitatively for positive vWF
staining, and was given a score of 1, 2 or 3 based on the size and
nature of the lesion as compared to the visual field at 20.times.
objective magnification. A further analysis using a confocal
microscope was performed to confirm the nature of the lesion if
needed.
The laser lesions were evaluated individually in a
semi-quantitative manner for positive staining of vWF and a score
was given to the size and to the nature of the laser lesion. The
lesion was characterized on whether the spot lesion was open and
had a central choroidal scar or was completely covered by RPE scar.
A grading of minimal (1) for presence of spot fluorescence, mild
(2) for presence of vWF-positive blood vessels/capillaries and
moderate (3) when the amount of blood vessels was more than average
in the areas of interest, center and periphery of the spot. The
presence of vWF-positive vessels was evaluated separately in the
center of the laser spot and around its periphery.
Blood Samples for Future Examination
The monkey blood was taken by femoral vein puncture: for tolerance
phase 1: before the beginning of the treatment and on days 1, 2, 3,
6 and 7; for efficacy phase 2: before the beginning of the
treatment and on days 1, 2, 3, 6, 12 and 28.
The samples were gently mixed and maintained under ambient
conditions until centrifugation, which was carried out as soon as
possible. The samples were centrifuged according to standard
procedures. The resulting serum was separated, transferred to
uniquely marked transparent polypropylene tubes and immediately
frozen on dry ice and transferred to a -80.degree. C. freezer. The
possible subsequent examinations include measurement of the
anti-CD160 antibody concentration in the systemic compartment after
IVT injection.
Terminal Procedure
Animals surviving until scheduled euthanasia were fasted overnight
before their scheduled necropsy. Prior to transportation from the
animal room to the necropsy area, a sedative (Ketamine HCl for
Injection, U.S.P.) were administered by intramuscular injection.
Animals underwent exsanguination by incision of the axillary or
femoral arteries following anaesthesia by intravenous injection of
sodium pentobarbital.
Tolerance to the IVT Injection of the Anti-hCD160 in Two Formats
(H7 IgG4 (ELB01101) and H7 IgG1 N297Q (ELB01111)) in the Cynomolgus
Eye.
Topical antibiotics (tobramycin at 0.3%) were applied to the two
eyes twice on the day before the treatment, after the injection and
twice on the day following the injection.
Before the dosage regimen, the phase-1 animals for evaluating
tolerance received an intramuscular injection of a sedative
cocktail of ketamine (5 mg/kg) and of dexmedetomidine (0.01 mg/kg)
followed by an isoflurane/oxygen mixture through a mask, judged
necessary to maintain the anaesthesia. After completion of the
dosage procedure (if judged to be necessary), the animals received
an intramuscular injection of 0.1 mg/kg of atipamezole, which is a
reversal agent for dexmedetomidine, if necessary.
During a first phase, the tolerance to the intravitreal (IVT)
injection of 1 mg of each of the anti-hCD160 H7 IgG4 and H7 IgG1
N297Q was verified by injection into 3 monkey eyes (H7 IgG4 in the
right eyes and H7 IgG1 N297Q in the left eyes).
The anti-CD160 H7 IgG4 antibody and the reference carrier control
were administered by a veterinarian-ophthalmologist to the
appropriate animals by bilateral intravitreal injection on day 1.
The target dose volume for each animal was 50 .mu.l/eye.
The phase-1 doses were administered using a 1 ml syringe and a
30-inch needle of 1/2 inch. During phase 1, H7 IgG4 was
administered into the right eyes and aglycosylated H7 IgG1 N297Q
was administered into the left eyes.
Demonstration of the Efficacy by the Preventive Effect on
Laser-Induced Choroidal Neovascularization of the IVT Injection of
H7 IgG4 Compared with the Carrier (PBS).
In two groups of 6 male cynomolgus monkeys (Macaca fascicularis)
(1.5 to 3.5 years old, weighing from 1.5 to 6 kg), the induction of
the choroidal neovascularization (CNV) was carried out as follows.
Before ophthalmic washing, mydriatic drops (benzalkonium chloride
(Zephiran.TM.)) were applied to each eye before any procedure.
The (efficacy) phase-2 animals were anaesthetized just like those
of the tolerance phase (see previous section).
The anti-CD160 H7 IgG4 antibody and the reference control carrier
were administered to the appropriate animals on day 1. On day 1,
they are injected by a veterinarian-ophthalmologist by bilateral
intravitreal IVT injection of 50 .mu.l at 20 mg/ml/eye of H7 IgG4
(the isoform selected after phase 1) or 50 .mu.l/eye of the
carrier. The target dose volume for each animal was 50 .mu.l/eye
with 1 mg of compound. The phase-2 doses were administered using an
Exelint U-100 0.5 cc insulin syringe with a needle of calibre
29.times.1/2 inch. A topical antibiotic was instilled in each
stated eye after the administration of the dose.
Procedure for Laser Induction of Choroidal Neovascularization
(ChNV)--Phase 2
On day 1 of phase 2, before the ChNV procedure, mydriatic drops
were applied to both the eyes. For the lesion laser-induction phase
or before the intravitreal (IVT) injections, the animals receive an
intramuscular injection of a cocktail of sedative ketamine (5
mg/kg), of glycopyrrolate (0.01 mg/kg) and of dexmedetomidine (0.01
mg/kg), and are then intubated with an endotracheal tube in order
to administer the isoflurane/oxygen mixture for maintaining the
anaesthesia, as was appropriate, and the animals were anaesthetized
with a cocktail of ketamine (5 mg/kg). After the dosage procedure
(as deemed necessary) is finished, the animals receive an
intramuscular injection of 0.1 mg/kg of atipamezole, which is a
reversal agent for dexmedetomidine, if deemed necessary. The
animals have also been divided up into treatment groups and
randomized by weight.
During the anaesthesia, on day 1, the laser treatment is carried
out by generating 9 lesions per eye concentrically with respect to
the fovea, with 1 lesion in the macular zone and 8 lesions in the
perimacular zone between the major vessels of the retina. The laser
lesions with an initial lesion size of 80 .mu.m were created using
an 810 nm diode laser at an initial power of 300 mW and a duration
of 0.1 sec. So, a total number of 108 laser sites/group was
evaluated for each treatment (6 animals/group, 2 eyes/animal, 9
sites/eye by items to be tested). The laser treatment was carried
out reproducibly and verified by the appearance of small bubbles of
vapour in the retina, characteristic of the rupture of the Bruch's
membrane. No lesion was directly generated on the fovea. The laser
parameters were adjusted as required in order to ensure rupture of
the Bruch's membrane (correlated with the bubble formation) and are
documented in the study data. All the notable events, such as
retinal haemorrhage, were documented for each laser lesion. The
eyes were kept hydrated with a saline and/or 1.0% sodium
carboxymethylcellulose solution during the procedure, if necessary.
Both the eyes were examined by slit-lamp biomicroscopy and/or
indirect ophthalmoscopy after each treatment had finished, in order
to confirm the position and the appearance of the dose and to
document any abnormality caused by the administration
procedure.
Analysis of Results
Tolerance Results, Summary:
Mortality and Clinical Signs
Clinical and ophthalmic examinations showed that there were no
treatment-related effects on hemorrhages or alteration of body
weights or macroscopic findings others than some that they were
considered as incidental or procedure-related and typical of
laboratory-housed primates. There was no effect linked to the
treatment on body weights or gains in body weight. There was no
effect linked to the treatment on body weight and there were no
macroscopic results. Very slight vitreal opacities were observed in
animals receiving H7 IgGl N297Q, on day 28. They were not
considered to be clinically important and such changes are commonly
observed using the intravitreal administration route.
Ocular Tolerance--Ophthalmological Observations
Some minor secondary observations were recorded at pretreatment;
however, all the animals were judged to be capable of taking part
in the study. During phase I, only minor changes were observed
after the administration of the dose. A small number of cells was
noted in the vitreous and anterior chamber in 3/6 eyes (No. 1002
and 1003).
In phase II, laser exposure resulted in similar procedure-related
ocular changes in all treated eyes, which included retinal
scarring, hemorrhages, and foveal hemorrhages. The chorioretinal
hemorrhages improved over time and had resolved in most eyes by day
28. Very slight cell-like opacities in the anterior portion of the
vitreous were noted on day 28 in 9 on 12 eyes given 1 mg H7 IgG4
(ELB01101). Control animal No. 2004 given D-PBS was also noted with
these cells bilaterally on days 9 and 28.
Efficacy of the IVT Injection of H7 IgG4 Assessed by the Preventive
Effect on Laser-Induced Choroidal Neovascularization
Analysis of the Fluoro-Angiography Results
On day 1, the eyes of all the animals were successfully subjected
to a scheme/design of 9 laser lesions for evaluating the choroidal
neovascularization (CNV). Although some animals were noted with
more than 9 lesions, only 9 lesions were evaluated.
As can be seen on the results summarized in FIG. 4 and below in
Table 18, when considering the number of clinically relevant
lesions (grade 3: moderate leakage and grade 4: substantial
leakage; combined), there was a greater number of these lesions in
the vehicle group on Days 14 and 29. At Day 14, there was a slight
effect of the of IgG4 H7 (ELB01101) in the reduction of ChNV
compared to the vehicle control. Following the evaluation of the
Day 29 lesions, the difference was more marked in favour of the
animals treated with the anti-CD160 H7 IgG4.
Indeed, on day 29, the animals receiving (ELB01101) exhibited a
smaller number (13) of clinically relevant lesions (grade 3:
moderate leakage and grade 4: significant leakage, combined)
compared with the vehicle (PBS) group (25). The incidence of
clinically relevant lesions relative to the total number of laser
lesions was 12% in the eyes administered with H7 IgG4 compared with
23.1% for the vehicle group. When considering the number of
clinically relevant lesions, there was a higher number of these
lesions in the PBS group on days 14 and 29.
As reported in Table 18, it should be noticed also that there were
a higher number of grade 0 lesions at day 14 and a higher number of
grade 1 and 2 at day 29 for ELB01101 than for the vehicle
indicating that in addition to a preventive effect on some large
lesions, for other minor lesions, ELB01101 seems to have delayed
the lesion progression.
TABLE-US-00018 TABLE 18 Impact of the vehicle control or H7 IgG4
treatments on the grades of the ChNV laser-induced lesions observed
in monkey eyes PBS number of H7 IgG4 number of Laser lesion lesions
(%/108 total lesions (%/108 total Day grades lesions evaluated)
lesions evaluated) 14 0 2 (1.9%) 11 (10.2%) 1 & 2 89 (82.4%) 86
(79.6%) 3 & 4 17 (15.7%) 11 (10.2%) 29 0 4 (3.7%) 4 (3.7%) 1
& 2 79 (73.1%) 91 (84.3%) 3 & 4 25 (23.1%) 13 (12.0%)
When incidence rate and incidence rate ratio are calculated for
this study (as defined in (Krzystolik et al., 2002)), at Day 29,
the incidence rate (IR) of clinically relevant lesions relative to
the total number of laser spots was 0.12 (13 out of 108) or 12% (in
percentage) in eyes administered with ELB01101 compared to 0.231
(25 out of 108) or 23.1% (in percentage) for the vehicle group,
corresponding to an incidence rate ratio (IRR) of 0.519
Evaluation of the Laser Lesions by von Willebrand Factor (vWF) by
Immunohistochemistry on NHP Tissues
The laser lesions were evaluated individually in a
semi-quantitative manner for positive staining of vWF and a score
was given to the size and to the nature of the laser lesion
A minimal classification was used
(1) for the presence of fluorescent labelling, slight
(2) for the presence of vWF-positive blood vessels/capillaries,
moderate
(3) when the amount of blood vessels was greater than the average
in the zones of interest, centre and periphery of the lesion.
The average score for vWF-positive staining was slightly higher
(approximately 10%) in the groups receiving H7 IgG4 at the centre
of the laser lesion, whereas the control group had a slightly
higher score (approximately 6%) at the periphery of the lesions
compared with the treatment alone.
Point Lesion Score
At a magnification of 20.times., the point of laser lesion received
a score of 1, 2 or 3 depending on the size of the lesion relative
to the visual field and the lesion was characterized in that the
point lesion was open, or had a central choroidal scar or was
completely covered with RPE scar.
An open lesion signifies that there is an absence of coverage of
the lesion by the Bruch's and RPE membranes. A choroidal scar is
characterized by the presence of dense tissue aggregates,
frequently at the centre of the site which appears fibrous and has
a high background fluorescence. Likewise, the RPE scar refers to an
altered conjunction of RPE cells or of aggregates with a central
fine fibrous structure with a high background fluorescence. Just as
with the vWF fluorescence results, the lesion size score in the
groups treated with H7 IgG4 was not very different from the
controls.
However, if the appearance of the lesion is considered by
evaluating the state of coverage of the lesion point by the RPE,
its opening and the presence or absence of choroidal scar, the
groups treated with H7 IgG4 had a higher total number of laser
lesions (32 to 16 lesions) completely covered by healing with RPE
membrane, whereas the control groups had a higher number of open
lesions with or without central scar (26 to 19 lesions) (Table 19).
This suggests a higher number of healing points in the groups
treated with H7 IgG4, on the condition that there was no difference
during the initial induction of the laser lesion. No recording was
made for animal No. 2101 because the integrity of the coverage of
the lesion was affected during the removal of the retina and of the
attached RPE membrane.
TABLE-US-00019 TABLE 19 Characterization of the laser lesions
present Group Saline control H7 IgG4 Total number of lesions per
animal Total No./ Total No./ 2101 2002 2003 2004 2005 2006 group
3001 3102 3003 3004 3005 3006 group Lesions covered 1 5 1 0 9 16 4
2 5 8 6 7 32 with healing by the RPE membrane Open lesions 2 2 5 8
0 17 1 4 1 0 0 2 7 Open lesions with 4 2 2 1 0 9 4 3 1 1 3 0 12
central choroidal healing
Summary:
As is seen in FIGS. 4 and 5 and in Tables 18 and 19, the efficacy
and the preventive effect on laser-induced choroidal
neovascularization of the IVT injection of H7 IgG4 compared with
the carrier (PBS) were demonstrated as follows.
On day 14, an effect of IgG4 H7 in the reduction of the choroidal
neovascularization compared with the PBS control was apparent. On
day 29, the difference was even more marked in the case of the
animals treated with H7 IgG4. On day 29, the incidence of
clinically significant lesions relative to the total number of
laser lesions was 12% in the eyes administered with H7 IgG4
compared with 23.1% for the PBS group. Taking into consideration
the combined number of clinically relevant lesions (grade 3:
moderate leakage and grade 4: significant leakage), there was a
greater number of these lesions in the PBS group on days 14 and 29
as can be seen in FIG. 4.
As is seen in FIG. 5, when evaluating the retinal pigment
epithelium (RPE) coverage status of each spot lesion using vWF IHC
(as shown in FIG. 5), spot sizes cores were not different between
ELB01101-treated and controls animals. The administration of H7
IgG4 was associated with a vascularization score that was slightly
higher at the centre of the laser lesion points, but slightly lower
at the periphery than in the control treatment, as shown by the
vWF-positive staining, and this correlates with the impact of H7
IgG4 on the clinically relevant lesions. In addition, there was a
higher incidence of laser lesions completely covered with RPE scar
compared with the control animals which, themselves, had an
increased incidence of "open" laser lesions with or without a
central choroid scar. These results suggest a process of
accelerated healing of the lesions in the animals receiving H7
IgG4, on the condition that the initial lesion created by the laser
is similar between the control animals and the treated animals. The
point size scores are not different between the animals treated
with IgG4 and the control animals.
In conclusion, the administration of H7 IgG4 (ELB01101) by single
bilateral intravitreal injection of 1 mg/eye was clinically
well-tolerated in cynomolgus monkeys. This was also the case with
the anti-CD160 H7 in the IgG1 N297Q format (ELB01111). H7 IgG4 was
associated with a reduction in choroidal neovascularization
compared with the carrier control (PBS), together with a slightly
higher vascularization at the centre of the induced lesion than at
the periphery and a higher incidence of lesions with healing of the
RPE membrane, which suggests a process of accelerated recovery in
the eyes treated with the anti-CD160 H7 IgG4 (ELB01101).
EXAMPLE 10
Tolerability and Dose Efficacy Assessment of Intravitreal
Injections of the Two Optimized Anti-CD160 mAb Formats (H7 Variant
D12 as an IgG4 (ELB01103) and as a Fab-Linker Fab Format
(ELB01132)) in the Laser-Induced (ChNV) Model in NHP
As described in example 9, one single intravitreal injection of 1
mg per eye of the non-optimized anti-CD160 IgG4 (ELB01101)
prevented by 50% the incidence of clinically relevant lesions
relative to the total number of laser spots without signs of major
ocular toxicity.
This first generation of anti-CD160 antibody was then optimized by
affinity maturation as described in example 2 and different mAbs
formats were designed as described in example 3. In example 8, the
different formats of anti CD160 designed for ophthalmological
purpose were compared in pharmacokinetic studies in rabbit and two
of them, ELB01103 (the affinity optimized anti CD160 as an IgG4)
and ELB01132 (the affinity matured as a Fab-linker-Fab) were
selected.
Compared to ELB01101, ELB01103 has a reduced systemic half-life and
still a good intravitreal half-life (4 days in rabbit). It should
have an increased or equivalent efficacy to ELB01101 due to its
affinity maturation. This affinity optimized anti CD160 was also
generated as a Fab-linker Fab format to increase eye permeability,
with a slightly reduced intravitreal half-life compared to ELB01103
and with a very shortened systemic half-life. The ELB01132 doses to
be tested were calculated based on an equimolar basis with
ELB01103. Indeed, the molecular mass of anti CD160 is roughly 150
KDa for the IgG ELB01103 and is reduced to 90 KDa for ELB01132.
The objectives of this study were: (1) to determine the
tolerability of two anti-CD160 formats at three doses (0.35, 1 and
3 mg per eye for ELB01103 and 0.25, 0.6 and 2 for ELB01132) when
given by a single bilateral intravitreal injection to the
cynomolgus monkey, and (2) to evaluate their potential preventive
effect on choroidal neovascularization in the laser-induced ChNV
monkey model.
The protocols used in this study for the follow up of animals and
for the assessment of tolerability and efficacy of test items
(ELB011's candidates) were those described in previous example 9,
with the following changes. The experimental design for the safety
and dose assessment for efficacy of each anti CD160 isoform is
described in Table 20 with 7 groups of 5 animals/group, male
only.
TABLE-US-00020 TABLE 20 Experimental design of dose efficacy NHP
experiment Experimental Design Dose Dose Level Concentration Number
of Group No. Test Material (mg/eye) (mg/mL) Males 1 Phosphate 0 0 5
Buffered Saline 2 ELB01103 0.35 7 5 3 (Anti-CD160 1 20 5 4 isoform
1) 3 60 5 5 ELB01132 0.21 4.14 5 6 Anti-CD160 0.59 11.83 5 7
isoform 2 1.8 35.5 5
The test and reference items described in Table 21 were
administered by bilateral intravitreal injection on day 0. The
target dose volume for each animal was 50 .mu.L/eye with the dose
of item to be tested. The doses were given using a 1 mL syringe and
an Exelint U-100 insulin 0.5 cc syringe with a 29
gauge.times.1/2-inch needle.
The batches of item that were tested are detailed in Table 21
below
TABLE-US-00021 TABLE 21 Items that were injected in dose efficacy
study Test and Reference Item Identification Reference Test Item
Test Item Item Identification ELB01103 ELB01132 Phosphate
(Anti-CD160 (Anti-CD160 buffered isoform 1) isoform 2) saline (PBS)
1X Initial Concentration 60 mg/mL 40 mg/mL and Cat#TMS-012-A, of
the sent batch 13.3 mg/mL Sigma and 4.6 mg/mL Aldrich-MERCK,
Volume, ml 1.2 mL 1.2 mL 250 mL Final concentrations 60 mg/mL and
35.5 mg/mL, that will be tested 20** mg/mL and 11.83** mg/mL 7**
mg/mL and 4.14** mg/mL Endotoxin level <0.5 EU/mL <0.5 EU/mL
<0.5 EU/mL (Endosafe .RTM.-PTS .TM.; <0.025 EU/mg <0.025
EU/mg Charles River) Storage conditions 2.degree. C. to 8.degree.
C. 2.degree. C. to 8.degree. C. 2.degree. C. to 8.degree. C.
**These solutions will be prepared by dilution of the items in PBS
1X
Laser-Induced Choroidal Neovascularization (ChNV) Procedure and
Evaluation of Active ChNV
Laser-induced ChNV procedure was the same as the one described in
Example 9. On day 1, for the evaluation of ChNV, the eyes of 5
animals per group were successfully subjected to a 9-spot laser
wound pattern between the major retinal vessels around the area of
the macula of each eye using a 810 nm diode laser at an initial
power setting of 300 mW, an initial spot size of 80 .mu.m and a
duration of 0.1 seconds. A total number of 90 laser sites/group was
evaluated for each treatment (5 animals/group, 2 eyes/animal, 9
sites/eye by items to be tested).
The development of active ChNV lesions was assessed by fluorescein
angiography (FA), once pre-study before injury and on days 14 and
28 after laser injury and the individual laser spots on the still
images were also evaluated for leakage semi quantitatively on a
scale of 0-4 by 2 independent readers with the same procedure as
previously described in example 9.
Spectral Domain-Optical Coherence Tomography (SD-OCT)
To assess impact of anti CD160 lead candidates on retinal thickness
at the lesion level site, there was an additional step of a
spectral domain optical coherence tomography (SD OCT) analysis of
the retina and of the different lesions sites. The SD-OCT analyses
were only performed on intermediate dose groups (1 mg for ELB01103
and 0.6 mg for ELB01132) and for the vehicle group on Days 14 and
28. For that, the pupils will be dilated using a mydriatic agent
(1% tropicamide and/or 2.5% phenylephrine). Animals will be
anesthetized for fluorescein angiography, as indicated in example
9. Serial image through laser lesion of 5 sections through each
lesion in both eyes were captured. The fibrovascular membrane area
were measured for each section and total volume calculated for each
spot. Measurement of retinal thickness in each lesion site compared
to three measures outside of the lesion (normal retinal thickness
evaluation). Additional scans or images were obtained, as deemed
necessary.
The evolution over time of individual retinal thickness of each
clinically relevant lesion could be followed as the mean change of
individual retinal thickness over time (between D14 and D28).
Efficacy of ELB011's candidates was then confirmed by looking at
the incidence of candidates on retinal thickness of each clinically
relevant lesion (grades 3+4) or of each grade 4 lesion and on
evolution of retinal thickness at the lesion site over time
(between D14 and D28).
Terminal Procedure and Bioanalysis (TK)
The terminal procedure for the animals was the same as the one
described in example 9. Animals will be subjected to a limited
necropsy examination, which will consist of an evaluation of the
tissues collected.
Sclera-choroid-RPE complex, vitreous and aqueous humors were
individually collected and kept frozen at -80.degree. c. until
analysis.
Some sera samples (0.75 mL) were collected for all animals using a
collect via the femoral vein over time (once pre study; 2, 6, 12,
24 and 48 hours post dose; Days 4, 7, 14 and 28). These sera
samples were kept at -80.degree. c. until the bioanalysis to assess
anti CD160 lead candidate concentration and or search and
quantification of any eventual anti-drug antibodies directed
against the anti CD160 leads.
Evaluation of Preventive and Therapeutic Efficacy of Lead
Candidates
In this study compared to the one described in example 9, there are
more analysed read outs to assess efficacy of these two anti CD160
lead candidates. Indeed, to assess efficacy of each anti CD160
isoform, first, their respective preventive effect was evaluated at
Day 14 and Day 28 on number and grade score of individual
clinically relevant laser induced lesions on ChNV area of
individual clinically relevant lesion and retinal thickness and,
second, their respective therapeutic effect was evaluated by
looking at the impact over time (between Day 14 and Day 28) on
established active lesions at D14 of mean grade score of clinically
relevant lesions, of individual and mean ChNV area (in pixel) of
clinically relevant lesions of individual and mean retinal
thickness (for the two intermediate doses (1 mg for ELB01103 and
0.6 mg for ELB01132) in function of the types of lesions, (all
grade, clinically relevant lesions (grade 3+4) and grade 4
only).
Results
The results of the ELB01103's and of ELB01132's profilings in terms
of efficacy and tolerability was performed in the NHP ChNV model
and are presented below.
ELB01103 and ELB01132 Safety Evaluation
As for ELB01101, and this whatever is the used ELB01103's or of
ELB01132's intravitreal dose, clinical and ophthalmic examinations
showed that there were no ELB01103 and ELB01132 treatment-related
effects on hemorrhages or alteration of body weights or macroscopic
findings others than some that they were considered as incidental
or procedure-related and typical of laboratory-housed primates.
Laser exposure resulted in similar procedure-related ocular changes
in all treated eyes, which included retinal scarring, hemorrhages,
and foveal hemorrhages. The chorioretinal hemorrhages improved over
time and had resolved in most eyes by Day 28.
Dose Efficacy of ELB01103 vs Vehicle Control and ELB01101 (H7 IgG4)
in NHP ChNV Model
As described in FIG. 6 panel A, the efficacy in a preventive
setting of H7 variant D12 as an IgG4 (ELB01103) was first evaluated
by looking at its incidence on clinically relevant lesions of high
grade associated with significant fluorescein leakage (grades 3+4)
over time (between Day 0 & Day 14 and Day 0 & Day 28).
There are several possibilities to present the efficacy data
obtained by semi quantitative evaluation of fluorescein
angiograms.
First, the efficacy data could be presented by the impact of the
tested item on percentage (%) of clinically relevant lesions/total
number of laser induced lesions as described in panel A FIG. 6.
This percentage corresponds to the number of clinically relevant
lesions divided by the total number of potential lesions (here 90)
for the 10 laser-induced monkey's eyes)*100. There is a clear
dose-dependent efficacy emerged at the two highest doses (1 mg and
3 mg) of ELB01103. The impact over the lesions seems stronger at
Day 28 than at Day 14 for groups 0.3 mg and 1 mg. At Day 14 maximal
effect was reached for the 3 mg dose. At Day 28, 1 mg ELB01103
(affinity matured mAb) has a greater efficacy than an equivalent
dose of ELB01101 (non-affinity matured H7 IgG4 candidate).
It is also possible to present the efficacy data looking at impact
on % of eyes with at least 1 grade 4 lesion or by the impact on %
of eyes with at least one clinically relevant lesions (grade 3+4).
A dose efficacy of ELB01103 was also observed when these read outs
are followed when one compared clinically relevant leakage (Grades
3/4) across groups and longitudinally (number of eyes or % of eyes
with at least one grade 4 lesion, data not shown).
Then, the impact of ELB01103 on evolution of the leakage severity
over time was assessed and this is represented in panel B of FIG.
6. The evolution of the leakage severity is seen by change over
time (Day 14 to Day 28) of grade score of individual clinically
relevant ChNV lesion. In panel B, when ELB01103 is injected
intravitreally, there is a mean reduction in leakage severity for
clinically individual lesions between Days 14 and 28, in particular
at the mid-dose (1 mg/eye), and at the low-dose (0.3 mg/eye).
Indeed, the mean change of grade score between Day 14 and Day 28 is
clearly reduced with these doses. For the 3 mg dose, it appears
that there is less control of leakage severity over time, but the
analysis is done on only 9 lesions that evolved between Day 14 and
Day 28 for the 3 mg dose due to a better antibody's efficacy at Day
14. However, the control of leakage severity for the 3 mg dose is
still better than for the control group.
Furthermore, the impact of dose escalating (0.3 to 3 mg) of
ELB01103 on mean change of ChNV area of clinically relevant lesions
from Day 14 to Day 28 is described in panel C of FIG. 6. The
measurement of lesion area showed that ELB01103-treated eyes were
comparable to controls on Day 14 and had lower leakage at all dose
levels when compared to the controls by Day 28. Indeed, while CHNV
area progressed over time in vehicle control group, the progression
of clinically relevant lesions that were not prevented at Day 14
was stopped between Day 14 and Day 28 when ELB01103 is added and
this with a dose-response effect. Whatever is the ELB01103 dose,
there is an important reduction of evolution of (individual data
not shown) and of mean ChNV area between Day 14 and Day 28 and this
with a dose response effect.
The impact of a dose of ELB01103 (1 mg) on mean change of retinal
thickness depending of lesion grade between Day 14 and Day 28 is
described in panel D of FIG. 6. While retinal thickness increased
over time in vehicle control group, the growth of clinically
relevant lesions followed by their retinal thickness was stopped
between Day 14 and Day 28 when ELB01103 is added. This reduction of
mean retinal thickness induced by ELB01103 between Day 14 and Day
28 is seen independently of the grade of the analysed lesion.
Dose Efficacy of ELB01132 vs Vehicle Control and ELB01101 (H7 IgG4)
in NHP ChNV Model
As described in FIG. 7 panel A, the efficacy in a preventive
setting of H7 variant D12 as a Fab linker Fab (ELB01132) was first
evaluated by looking at its incidence on clinically relevant
lesions of high grade associated with significant fluorescein
leakage (grades 3+4) over time. There is an efficacy of the two
lowest doses (0.23 mg and 0.6 mg). However, there is no ChNV lesion
prevention at all for the highest dose (2 mg). Consistently, the
intermediate dose (0.6 mg) of ELB01132 is very efficient to prevent
ChNV lesions appearance by Day 14 whatever is the observed read
outs of efficacy (data not shown). This is true when one compared
clinically relevant leakage (grades 3/4) across groups and
longitudinally (number of eyes or % of eyes with at least one grade
4 lesion data not shown). The ELB01132's 0.6 mg dose efficacy is
better than ELB01101's efficacy.
Then, the impact of ELB01132 on evolution of the leakage severity
over time was assessed and this is represented in panel B of FIG.
7. The evolution of the leakage severity is seen by change over
time (Day 14 to Day 28) of grade score of individual clinically
relevant ChNV lesion. In panel B of FIG. 7, when ELB01132 is
injected intravitreally, the ChNV lesions evolution was controlled
only at low dose. In contrast, when mid- and high doses are used,
there is an increased leakage. At intermediate dose, the control of
leakage severity over time for the 2 mg dose is represented but the
analysis is done on only 5 lesions due to antibody efficacy at Day
14. Regarding data obtained from the highest dose (2 mg), there is
no control at all of ELB01132 on leakage at this dose.
Furthermore, the impact of dose escalating (0.25 to 2 mg) of
ELB01132 on mean change of ChNV area of clinically relevant lesions
from Day 14 to Day 28 is described in panel C of FIG. 7.
In eyes given ELB01132, smaller lesion areas were observed at the
mid dose (0.6 mg/eye) on Days 14 and 28, when compared to the
control eyes. Between Days 14 and 28, leakage development was only
slightly lower than in the control eyes. Only the two highest doses
tend to slightly reduce the increase evolution of mean ChNV area
between Day 14 and Day 28.
The impact of a 0.6 mg dose of ELB01132 on mean change of retinal
thickness of several types of lesions over time is described in
panel D of FIG. 7. While retinal thickness increased between Day 14
and Day 28 in vehicle control group, the control of the 0.6 mg dose
of ELB01132 on change over time of mean retinal thickness of
lesions is much variable depending of lesions grade than for
ELB01103. Indeed, when clinically relevant lesions of grades 3 and
4 are considered, the mean change of retinal thickness is
drastically reduced with 0.6 mg ELB01132, but, this is not the case
anymore when all grades of lesions or when only the grade 4 lesions
are considered independently (see FIG. 14). However, in the case of
the analysis of the grade 4 lesions the analysis is done on only 5
grade 4 lesions due to antibody efficacy at Day 14.
Conclusion
Administration of two isoforms of H7 variant D12 anti-CD160 by
single bilateral intravitreal injection was clinically
well-tolerated in cynomolgus monkeys at up to 3 mg ELB01103/eye and
2 mg ELB01132/eye. Both test items were associated with a reduction
of ChNV progression, as measured by change in clinically relevant
lesion area and/or thickness, when compared to the control. In
general, the efficacy of ELB01103 was higher than observed for
ELB01132. Indeed, there is a clear dose effect of the ELB01103
whatever is the analyzed efficacy read out while ELB011032 efficacy
is much variable in function of dose and in function of efficacy
read out. However, consistently, the intermediate dose (0.6 mg) of
ELB01132 is very efficient to prevent ChNV lesions appearance.
EXAMPLE 11
Binding of the H7 IgG1 Antibody on Tumour Cells of CLL Patients
The PBMCs isolated from 7 CLL patients were labelled with the
antibodies CL1-R2 (murine anti-CD160 IgG1), anti-CD160 H7 in IgG1
format, or BY55 (murine anti-CD160 IgM), in a CD19/CD5/CD3/CD56
panel (see FIG. 8). The CD5+CD19+ tumour cells were analysed in
order to measure the fluorescence intensity of the CD160 labelling.
CD160 expression is detectable on all the CLL samples with variable
intensities. As can be seen in FIG. 8, the H7 IgG1 antibody binds
efficiently to the tumour cells in 6/7 of the CLL samples examined
and this better than CL1-R2 or commercial BY55 anti CD160 mAbs.
The H7 antibody in the IgG1 format is thus capable of binding to
the tumour cells in the CLL, and can thus be used to target and
kill these malignant cells by a cytotoxicity mechanism such as in
particular ADCC or CDC.
EXAMPLE 12
In Vitro Evaluation of ADCC Induced by the H7 Antibody in the IgG1
Format on CD160-Positive Cells
The anti-CD160 antibody H7 in the IgG1 format kills cells
expressing CD160 by a mechanism of ADCC (see FIG. 9).
NK cells purified from the blood of a healthy donor were used as
effectors in a test measuring the ADCC activity of the anti-CD160
H7 IgG1 antibody. The E300-CD160 target cells (transfected pre-B
human cell line expressing CD160) were labelled with CFSE and
incubated with the effector NK cells in the presence of the H7 IgG1
antibody or of a human IgG1 isotype control, at the effector/target
ratios indicated (1/1, 1/5 and 1/10). The percentages of target
cells killed were measured by labelling with 7AAD and flow
cytometry analysis. The percentages of doubly labelled 7AAD+CFSE+
dead cells are indicated in the top right quadrant on the dot-plots
presented.
These results and also those presented in FIG. 8 (Example 11) show
that the H7 antibody in the IgG1 format can be used to target and
kill cells expressing CD160 at their surface, by a mechanism of
ADCC.
EXAMPLE 13
Activation of NK Cells and of Their Production of Interferon-Gamma
by the H7 Antibody in the IgG.sub.1 Format
As shown by the results of FIGS. 10, 11 and 12, the anti-CD160 H7
antibody in the IgG1 format activates NK cells.
As shown in FIG. 10, panel A, the H7 IgG1 antibody is capable of
binding to the surface of human NK cells purified from peripheral
blood.
FIG. 10, panel B, shows that H7 IgG1 induces the production of
interferon-gamma (IFN-.gamma.) by NK cells. NK cells purified from
the blood of a healthy donor were cultured for 24 h in wells of
96-well plates (1.times.10.sup.+6 cells per well) alone or in the
presence of the H7 IgG1 antibody, or of a human IgG1 isotype
control, concentrated to 1 or 10 .mu.g/ml. The IFN-.gamma. was
assayed by ELISA in the culture supernatants. The results presented
are means of triplicates+/-sem.
FIG. 10, panel C shows that H7 IgG1 induces the expression of the
CD69 activation marker on NK cells. In the same experiment as in
panel B, the NK cells were collected after 24 h of culture and
labelled with an anti-CD69 antibody conjugated to the fluorochrome
APC. The percentages of CD69-positive cells were analysed by flow
cytometry. The means(+/-sem) were calculated from triplicates.
The anti-CD160 H7 antibody in the IgG1 format, but not in the IgG4
format, activates NK cells, as shown in FIG. 11. NK cells purified
from the blood of a healthy donor were cultured alone or in the
presence of the following antibodies concentrated to 5 .mu.g/ml: H7
IgG1, H7 IgG4, their respective human IgG1 or IgG4 isotype
controls, or the antibodies ELB01103, ELB01104 and ELB01106, which
are variants derived from the H7 antibody in the IgG4 format. All
the antibodies were controlled to verify the absence of
contamination by endotoxins. The anti-CD16 antibody (ebioscience
cat #16-0166) is used as positive control. The NK cells
(5.times.10.sup.+5 per well) were collected after 24 h of culture
and labelled with an anti-CD69 antibody conjugated to the
fluorochrome APC. The percentages of CD69-positive cells were
analysed by flow cytometry (means of triplicates+/-SD). The
anti-CD160 H7 in the IgG1 format induces the expression of the CD69
activation marker on NK cells, whereas the same antibody in the
IgG4 format has no effect. The variants of H7 in the human IgG4
format (ELB01103, ELB01104 and ELB01106) also do not exhibit any
activating effect on NK cells.
EXAMPLE 14
Increased NK Cell-Stimulating Activity with the Different Variants
of H7 in the IgG1 and E345K/IgG1 Formats
As shown by the results of FIG. 12: The variants derived from the
anti-CD160 H7 antibody in the IgG1 and E345K/IgG1 formats have an
increased capacity to activate NK cells. NK cells purified from the
blood of a healthy donor were cultured for 24 h in wells of 96-well
plates (1.times.10.sup.+6 cells per well), alone or in the presence
of the anti-CD160 H7 IgG1 antibody, or of the variants ELB02102,
ELB02103, ELB02104 (all three in the IgG1 format), ELB02112,
ELB02113 or ELB02114 (all three in the E345K/IgG1 format) produced
by ElsaLys, at doses of 0.001 to 10 .mu.g/ml. A human IgG1 at 10
.mu.g/ml was used as negative isotype control, and an anti-CD16
(ebioscience cat #16-0166) was used as positive control.
The IFN-gamma was assayed by ELISA in the culture supernatants. The
results presented are means of triplicates+/-sem.
The NK cells were collected and labelled with an anti-CD69 antibody
conjugated to the fluorochrome APC. The percentages of
CD69-positive cells were analysed by flow cytometry. The
means(+/-sem) were calculated from triplicates.
These results were analysed using the GraphPad Prism software for
generating non-linear regression curves (Log(agonist) vs response,
3-parameter equations) and calculating the median effective
concentrations (EC50s). The EC50s for induction of CD69 were not
calculated for the 3 variants ELB02112, ELB02113 or ELB02114,
because of the mortality observed with the concentrations greater
than or equal to 0.1 .mu.g/ml. The mortality of the NKs stimulated
by these E345K/IgG1 formats is probably induced following the
strong activation of the cells.
All of these results show that the three variants of H7 in the IgG1
format (ELB02102, ELB02103, ELB02104) are much more potent than the
original H7 IgG1 antibody (ELB02101) for activating NK cells, with
an improvement of 2 to 3 logs in the EC50s.
The three variants of H7 in the E345K/IgG1 format (that is to say
ELB02112, ELB02113, ELB02114) exhibit a further increased capacity
for inducing IFN-gamma production, with an additional improvement
of 2 logs in the EC50s (4 logs relative to the original H7 IgG1
antibody (ELB02101)).
The results presented in Examples 13 and 14 show that the H7
antibodies and the variants thereof in the IgG1 and E345K/IgG1
formats are capable of activating NK cells and of inducing their
IFN-.gamma. production. These properties make them capable of
stimulating the immune response in patients suffering from cancer,
via NK cells, and indirectly via T lymphocytes and
antigen-presenting cells activated by IFN-.gamma., a cytokine known
to activate Th1-type responses.
Furthermore, these properties make the antibodies derived from H7
in the IgG1 and E345K/IgG1 formats potentially capable of
increasing the ADCC cytotoxic activity induced by other antibodies
possessing this mode of action that would be co-administered, and
thus would make it possible to improve their therapeutic
effects.
EXAMPLE 15
Labelling of NK and CD8+ T Cells by the Variants of the H7 Antibody
in the IgG1 and E345K/IgG1 Formats
The variants derived from the anti-CD160 H7 antibody in the IgG1
and E345K/IgG1 formats label NK and CD8+ T cells more efficiently
(FIG. 13).
The PBMCs (peripheral blood mononuclear cells) of two healthy
donors were analysed by flow cytometry after immunolabelling with
anti-CD45, CD3, CD4, CD8 and CD19 antibodies and with the
anti-CD160 antibodies indicated conjugated to PE (Lynx Rapid RPE
Antibody Conjugation Kit ref LNK022RPE) (0.25 .mu.g for
5.times.10.sup.+5 PBMCs). An irrelevant human IgG1 (hIgG1) was used
as negative control, the Fc receptors were saturated with a human
Fc (Rockland), 15 min AT.
In FIG. 13, panel A: The variants of H7 in the IgG1 format
(ELB02102, ELB02103, ELB02104) or E345K/IgG1 format (ELB02112,
ELB02113, ELB02114) bind more efficiently to NK cells than the
original H7 IgG1 antibody, with 60% to 80% of NK cells positively
labelled. In FIG. 13, panel B: A population of CD8+ T cells,
clearly detected in donor 2, is also labelled more efficiently with
the H7 variants.
These results show that the variants derived from the anti-CD160 H7
antibody in the IgG1 and E345K/IgG1 formats bind to NK and CD8+ T
cells more efficiently than the original H7 IgG1 antibody.
These results and those presented in the previous examples show
that the H7 IgG1 antibody and the variants thereof in the IgG1 and
E345K/IgG1 formats can bind not only to NK cells and stimulate
their activity, but also to a population of CD8.sup.+CD160.sup.+ T
lymphocytes, the activity of which they could also modulate.
EXAMPLE 16
Design and Generation of Bispecific Antibodies (bsabs) for the
Anti-CD160 Candidates, Optimized for Ophthalmology and Oncology
Possible Bispecifics (bsabs) with an Anti-CD160 for Ophthalmology
Indications: Potential Second Valency to be Combined with an
Anti-CD160 H7 or Affinity Matured.
The strategy described by (Labrijn et al., 2014) was applied for
developing a bispecific IgG from the parental antibodies
anti-hCD160 IgG1 F405L (clone H7) or a derivative thereof) and IgG1
K409R consisting of an anti hAngiopoietin 2 as proof of
concept.
One of the antibodies is chosen from Table 22 (for the
ophthalmology application) or from Table 23 (for the oncology
application). This first antibody is in the IgG1 N297Q H310A-H435Q
K409R format for ophthalmology or IgG1 K409R format for oncology.
The second antibody is the anti-CD160 H7 (or a variant thereof).
This second antibody is in the IgG1 N297Q H310A-H435Q F405L format
for the ophthalmic indications and in the IgG1 F405L format for the
oncology.
TABLE-US-00022 TABLE 22 Antibody targeting antigens that can be
used in ophthalmology as second potential valency for producing an
anti- CD160 bsab or to be used in combination therapy with an
anti-CD160 H7 or a variant thereof. Antibody against bsab or human
antigen combination targeted therapy Rationale Anti- Combination
Inhibits neo-angiogenesis angiopoietin 2 therapy or bsab Pathway
blocker Inhibits TEMs and inflammation Anti-CD200R Combination
Inhibits TEMs and inflammation (agonist) therapy or bsab Inhibits
VEGF secretion by macrophages (inhibition of the neo-angiogenesis?)
Anti-angiopoietin Combination Inhibits neo-angiogenesis by like 4
therapy or bsab targeting anti-apoptotic signals No impact on
inflammation Anti-PDGF BB Combination Inhibits pericyte
stabilization therapy or bsab Anti-VEGF Combination Inhibits
neo-angiogenesis by (avastin or targeting pro-angiogenic factors
lucentis) Anti-beta Combination Inhibits inflammation amyloid
therapy or bsab Anti-PS Combination Inhibits neo-angiogenesis by
therapy or bsab targeting abnormal vascularization
Anti-sphingosine-1- Combination Inhibits neo-angiogenesis by
phosphate therapy or bsab targeting abnormal vascularization
Anti-C'5 Combination Inhibits inflammation therapy or bsab
Anti-CD115 Combination M1/M2 Polarization therapy
TABLE-US-00023 TABLE 23 Antibody targeting antigens that can be
used in oncology as potential second valency for producing an
anti-CD160 bsab or to be used in combination therapy with an
anti-CD160 H7 or a variant thereof. Antibody against bsab or human
antigen combination targeted therapy Rationale Anti-Ang2
Combination Inhibits neo-angiogenesis therapy Inhibits TEMs and
inflammation or bsab Anti-CD200R Combination Inhibits TEMs and
inflammation therapy Inhibits VEGF secretion by or bsab macrophages
(inhibition of neo- angiogenesis?) Anti-CD19 Combination
Simultaneous targeting of two or -CD20 therapy antigens on CLLs for
increasing or bsab tumour specificity and efficacy of B CLL cell
lysis. Anti-CD200 as Combination Simultaneous targeting of two CLL
TAA taa therapy antigens on CLLs for increasing or bsab tumour
specificity and efficacy of B CLL cell lysis. Anti-CD180
Combination Simultaneous targeting of two therapy antigens on CLLs
and on marginal or bsab zone lymphomas (MZL) for increasing tumour
specificity and the efficacy of B lymphoma lysis. Anti-CD148
Combination Simultaneous targeting of two therapy antigens on CLLs
and on mantel or bsab cell tumours (MCL) for increasing tumour
specificity and the efficacy of B lymphoma lysis. Anti-CD47
Combination Increase of ADCP in addition therapy to ADCC or
bsab
This technology can be used to generate the anti-CD160 bsab
candidates according to the targeted ophthalmology, oncology or
immunotherapy indications.
EXAMPLE 17
Evaluation of the Anti-hCD160/Anti-Human Angiopoietin 2 or
Anti-hCD160/Anti-Human CD200R Combination Therapies and
Bispecifics
The combination therapies of anti-CD160 and its anti-X partner
antibody (in particular where X is angiopoietin 2 or CD200R) and
anti-CD160/anti-X bispecifics are evaluated for their efficacy and
for the additivity and/or the synergy of their efficacy in the
rabbit model of corneal neovascularization induced in sodium
hydroxide (NaOH) buffer as described in (Campos-Mollo et al.,
2011), at two doses of each antibody (100 and 500 .mu.g) or 100 and
500 .mu.g of the bsab.
EXAMPLE 18
Antibodies According to the Invention are Able to Bind on CD160 GPI
and CD160 TM, Although CL1-R2 Antibody Recognizes Only CD160
GPI>>
The binding capacity of the anti-CD160 CL1-R2, ELB02101 (H7 IgG1)
antibodies and of the H7 variants in the ELB02104, ELB02114 and
ELB01103 formats was evaluated during the labelling of surface
human CD160-GPI (glycosylphosphatidylinositol), expressed in a
recombinant cell line CHO-S-hCD160-GPI (clone 2G10) and during the
labelling of surface human CD160-TM (transmembrane), expressed in a
recombinant cell line CHO-S-hCD160-TM in comparison with
non-transfected CHO-S cells, by measuring the percentage of cell
labelled=percentage of binding (see FIG. 14). For this,
2.times.10.sup.+5 CHO-S-hCD160-GPI, CHO-S-hCD160-TM and
non-transfected CHO-S cells were labelled with 1 .mu.g of each of
these antibodies and also with the appropriate control
isotypes.
In FIG. 14, all the anti-CD160 tested (regardless of the isotype or
the IgG format or the variant) specifically recognize human
CD160-GPI expressed recombinantly by the CHO-S cells. Humanized
ELB02101 (H7 IgG1) and the H7 variants in their different formats
ELB02104, ELB02114 and ELB01103 bind more efficiently to the
CHO-hCD160-GPI transfectants than the parental CL1-R2. Unexpectedly
humanized ELB02101 (H7 IgG1) and the variants ELB02104, ELB02114
and ELB01103 bind also to human CD160-TM expressed recombinantly by
the CHO-S cells while parental CL1-R2 mAb is not able to do that
whatever is the tested dose.
EXAMPLE 19
T CD4 Re-Activation Through the Blocking of HVEM-CD160 Interaction
by the H7 Variant: ELB02104
As shown by the results of FIG. 15, the A09 variant derived from
the anti-CD160 H7 antibody in the IgG1 format (ELB02104) is able to
re-activate T CD4 CD45.sup.High CD160+ lymphocytes compare to
control isotype. Through the blocking of HVEM-CD160 interaction,
ELB02104 removes the inhibition of TCD4 cells induced by HVEM
protein.
T CD4 lymphocytes cells purified from the blood of a healthy donor
were cultured for 16 h in 96-well plates (1.times.10.sup.+6 cells
per well), in the presence of the anti-CD160 mAb: ELB02104 or with
the appropriate control isotype at 10 .mu.g/ml and with anti-CD3
(clone UTCH1) mAb+/-anti-CD28 (clone CD28.2) mAb+/-HVEM protein
(10334-H08H, Sino biological) coated on the plate.
T CD4 lymphocytes were collected and labelled with a viability
marker: Zombie NIR, an anti-CD45RA antibody conjugated to the
fluorochrome BB515 targeting Naive/Memory cells, with an anti-CD160
(clone BY55) antibody conjugated to the fluorochrome Alexa fluor
647 targeting CD160 expressing cells and with an anti-CD69 antibody
conjugated to the fluorochrome PE targeting activated cells. The
percentage of Zombie NIR-/CD45RA.sup.high+/CD160+/CD69+ positive
cells were analysed by flow cytometry. The means(+/-sem) were
calculated from duplicates. Results show that the H7 A09 variant in
the IgG1 format (ELB02104) blocks HVEM-CD160 interaction and
removes the inhibition of TCD4 cells induced by HVEM protein as
shown by the upregulation of CD69, an activation marker express by
T CD4 CD45RA.sup.High CD160+ cells.
HVEM is expressed by several cancers and play a role in tumor
progression and immune evasion. Blocking HVEM-CD160 axis on T CD4
cells may restore anti-tumoral responses by T CD8 cytotoxic
generation.
EXAMPLE 20
DC (Dendritic Cells) Maturation Through NK Cell-Stimulating
Activity of H7 IgG1 (ELB02101) and of H7 A09 in the IgG1 (ELB02104)
and E345K/IgG1 (ELB02114) Formats
The H7 A09 variant antibody in the IgG1 (ELB02104) and E345K/IgG1
(ELB02114) formats have an increased capacity to induce DC
maturation cells through NK cell-stimulating activity compare to H7
IgG1 (ELB02101) and contrary to parental CL1-R2 mAb.
Monocytes cells purified from the blood of a healthy donor were
differentiated in immature dendritic cells with GM-CSF (100 ng/mL)
and IL-4 (20 ng/mL) for 6 days. NK cells purified from the blood of
a healthy donor were cultured for 16 h in 96-well plates
(1.times.10.sup.+6 cells per well), alone or in the presence of the
anti-CD160 mAbs: CL1-R2, H7 IgG1 (ELB02101), or of the variants
ELB02104 (in the IgG1 format), ELB02114 (in the E345K/IgG1 format)
or ELB01103 (in the IgG4 format), at 10 .mu.g/ml and also with the
appropriate control isotypes. Immature DC (1.times.10.sup.+5 cells
per well) were coculture with NK previously incubated with
anti-CD160 mAbs 24 h.
The DC/NK co-culture cells were collected and labelled with a
viability marker: Zombie NIR, an anti-CD11c antibody conjugated to
the fluorochrome PE-Cy7 targeting DC cells, with an anti-CD56
antibody conjugated to the fluorochrome Viobright 515 targeting NK
cells and with an anti-CD86 antibody conjugated to the fluorochrome
BV421 targeting mature DC cells. The percentage of Zombie
NIR-/CD11c+/CD86+ positive cells were analyzed by flow cytometry.
The means(+/-sem) were calculated from duplicates.
Results show that H7 A09 variant in the IgG1 (ELB02104) and in the
E345K/IgG1 (ELB02114) formats have an increased capacity to induce
DC maturation cells (as shown by CD86 upregulation) through NK
cell-stimulating activity compare to H7 IgG1 (ELB02101).
Surprisingly parental CL1R2 mAb has not this property to induce DC
maturation.
The results presented in examples 13 and 14 show that the H7
antibodies and the variants thereof in the IgG1 and E345K/IgG1
formats are capable of activating NK cells and inducing their
IFN-.gamma. production. These properties make them capable of
stimulating DC maturation and indirectly via the cross talk between
mature DC and T lymphocytes to drive the generation of cytotoxic T
lymphocytes with anti-tumoral properties in cancer patients.
BIBLIOGRAPHY
Giustiniani et al., J Immunol. 2009 Jan. 1;
182(1):63-71--Identification and characterization of a
transmembrane isoform of CD160 (CD160-TM), a unique activating
receptor selectively expressed upon human NK cell activation
El-Far et al., J Transl Med. 2014 Sep. 2; 12:217. doi:
10.1186/s12967-014-0217-y. CD160 isoforms and regulation of CD4 and
CD8 T-cell responses.
E A Kabat, T T Wu, C Foeller, H M Perry, K S Gottesman (1991)
Sequences of Proteins of Immunological Interest
Diebolder et al., Science. 2014 Mar. 14; 343(6176):1260-3. doi:
10.1126/science.1248943--Complement is activated by IgG hexamers
assembled at the cell surface. Diebolder C A
de Jong et al., Published online 2016 Jan. 6. doi:
10.1371/journal.pbio.1002344--A Novel Platform for the Potentiation
of Therapeutic Antibodies Based on Antigen-Dependent Formation of
IgG Hexamers at the Cell Surface
Wang et al., Mol. Cell. 2016 Jul. 7; 63(1):135-45. doi:
10.1016/j.molcel.2016.05.016--Molecular Basis of Assembly and
Activation of Complement Component C1 in Complex with
Immunoglobulin G1 and Antigen.
Krzystolik et al., Arch Ophthalmol. 2002 March;
120(3):338-46--Prevention of experimental choroidal
neovascularization with intravitreal anti-vascular endothelial
growth factor antibody fragment.
Gadkar et al., Invest Ophthalmol Vis Sci. 2015 August;
56(9):5390-400. doi: 10.1167/iovs.15-17108--Design and
Pharmacokinetic Characterization of Novel Antibody Formats for
Ocular Therapeutics
Labrijn et al., Nat Protoc. 2014 October; 9(10):2450-63. doi:
10.1038/nprot.2014.169. Epub 2014 Sep. 25. --Controlled Fab-arm
exchange for the generation of stable bispecific IgG1
SEQUENCE LISTINGS
1
641121PRTMus musculus 1Gln Val His Leu Gln Gln Ser Gly Ala Glu Leu
Ala Arg Pro Gly Ala1 5 10 15Ser Val Lys Leu Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Tyr 20 25 30Trp Met Gln Trp Ile Lys Gln Arg Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ser Ile Tyr Pro Gly Asp Asp
Asp Ala Arg Tyr Thr Gln Lys Phe 50 55 60Arg Gly Lys Ala Thr Leu Thr
Ala Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75 80Met Gln Leu Ser Ser
Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly
Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly
Thr Thr Leu Thr Val Ser Ser 115 1202107PRTMus musculus 2Asp Ile Val
Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly1 5 10 15Asn Ser
Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn His 20 25 30Leu
His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40
45Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu
Thr65 70 75 80Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser
Trp Pro Leu 85 90 95Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
105310PRTMus musculus 3Gly Tyr Thr Phe Thr Asp Tyr Trp Met Gln1 5
10416PRTMus musculus 4Ser Ile Tyr Pro Gly Asp Asp Asp Ala Arg Tyr
Thr Gln Lys Phe Arg1 5 10 15512PRTMus musculus 5Arg Gly Ile Ala Ala
Val Val Gly Gly Phe Asp Tyr1 5 10611PRTMus musculus 6Arg Ala Ser
Gln Ser Ile Ser Asn His Leu His1 5 1077PRTmurine retrovirus Cas
NS-1 7Tyr Ala Ser Gln Ser Ile Ser1 589PRTMus musculus 8Gln Gln Ser
Asn Ser Trp Pro Leu Thr1 59447PRTHomo sapiens 9Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Phe 20 25 30Trp Met Gln
Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala
Ile Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr Gln Lys Phe 50 55 60Arg
Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ala Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440
44510447PRTHomo sapiens 10Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Ser Asp Tyr 20 25 30Trp Met Gln Trp Ile Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp
Asp Asp Ala Arg Tyr Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg
Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln
Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215 220Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu Ala Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn Gln Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu Gly 435 440 44511121PRTArtificial
Sequencehumanized sequence 11Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Trp Met Gln Trp Ile Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ser Ile Tyr Pro Gly
Asp Asp Asp Ala Arg Tyr Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr
Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Leu Thr Val Ser Ser 115 12012107PRTArtificial
Sequencehumanized sequence 12Asp Ile Gln Leu Thr Gln Ser Pro Ser
Phe Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Asn His 20 25 30Leu His Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser
Ile Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp Phe
Ala Thr Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys 100 10513443PRTHomo sapiens
13Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp
Tyr 20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Arg Tyr Thr
Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val
Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val
Ser Ser Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155
160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser 180 185 190Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly Pro Pro Cys Pro 210 215 220Pro Cys Pro Ala Pro Glu Phe Leu
Gly Gly Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys
Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280
285Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val305 310 315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Gln 340 345 350Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser385 390 395
400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435
44014107PRTArtificial Sequencehumanized sequence 14Glu Ile Val Leu
Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn His 20 25 30Leu His
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Lys
Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65
70 75 80Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro
Leu 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100
10515326PRTHomo sapiens 15Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys Asn
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110Glu
Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120
125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr
Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235
240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu
Gly 32516329PRTHomo sapiens 16Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
3251798PRTHomo sapiens 17Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val1819PRTArtificial Sequencepeptide signal 18Met Glu Trp Ser Trp
Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly1 5 10 15Val His
Ser1920PRTArtificial Sequencepeptide signal 19Met Asp Trp Pro Val
His Leu Leu Val Leu Leu Leu Phe Trp Ile Pro1 5 10 15Gly Ala Met Ala
202020PRTArtificial Sequencepeptide signal 20Met Ser Val Pro Thr
Gln Val Leu Gly Leu Leu Leu Leu Trp Leu Thr1 5 10 15Asp Ala Arg Cys
202120PRTArtificial Sequencepeptide signal 21Met Asp Trp Pro Val
His Leu Leu Val Leu Leu Leu Phe Trp Ile Pro1 5 10 15Gly Val Arg Ala
2022107PRTHomo sapiens 22Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys 100 10523107PRTHomo sapiens 23Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu1 5 10
15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Val Leu
Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu65 70 75 80Lys His Lys Leu Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 100 10524107PRTHomo sapiens 24Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His
Lys Leu Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10525121PRTHomo sapiens
25Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp
Tyr 20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Arg Tyr Thr
Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val
Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val
Ser Ser 115 12026121PRTHomo sapiens 26Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Ser Glu Ser 20 25 30Trp Met Gln Trp Ile
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr
Pro Gly Asp Asp Glu Ala Arg Val Thr Gln Lys Phe 50 55 60Arg Gly Arg
Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly
100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser 115 12027121PRTHomo
sapiens 27Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asp Tyr 20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Gln
Phe Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala
Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu
Thr Val Ser Ser 115 12028121PRTHomo sapiens 28Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr 20 25 30Trp Met Gln
Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala
Ile Tyr Pro Gly Asp Asp Asp Ala Gln Tyr Thr Gln Lys Phe 50 55 60Arg
Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser 115
12029121PRTHomo sapiens 29Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Ser Asp Phe 20 25 30Trp Met Gln Trp Ile Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp
Asp Asp Ala Arg Val Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg
Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln
Gly Thr Thr Leu Thr Val Ser Ser 115 12030121PRTHomo sapiens 30Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Phe
20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr Gln
Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly
Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser
Ser 115 12031326PRTHomo sapiens 31Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105
110Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230
235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 275 280 285Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser
Leu Gly 32532326PRTHomo sapiens 32Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105
110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu Ala Gln Asp 180 185 190Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230
235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 275 280 285Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala
Leu His Asn Gln Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser
Leu Gly 32533326PRTHomo sapiens 33Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105
110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125Asp Thr Leu Met Ala Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe 165 170 175Gln Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230
235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 275 280 285Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser
Leu Gly 32534329PRTHoJo virus 34Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Gln Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190Ala Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn Gln Tyr Thr305 310 315 320Gln
Lys Ser Leu Ser Leu Ser Pro Gly 32535329PRTHomo sapiens 35Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met
Ala Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32536229PRTHomo sapiens 36Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Trp Met Gln Trp Ile Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ser Ile Tyr Pro Gly Asp
Asp Asp Ala Arg Tyr Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg
Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln
Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr
His Thr22537219PRTHomo sapiens 37Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Ser Asp Phe 20 25 30Trp Met Gln Trp Ile Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro
Gly Asp Asp Asp Ala Arg Val Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala
Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val 210 21538235PRTHomo sapiens 38Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Phe
20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr Gln
Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly
Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala225 230 2353915PRTHomo sapiens 39Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser1 5 10 1540467PRTHomo sapiens 40Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25
30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45Gly Ser Ile Tyr Pro Gly Asp Asp Asp Ala Arg Tyr Thr Gln Lys
Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly
Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly225 230 235 240Ser Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly 245 250 255Ala Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp 260 265 270Tyr Trp Met
Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 275 280 285Ile
Gly Ser Ile Tyr Pro Gly Asp Asp Asp Ala Arg Tyr Thr Gln Lys 290 295
300Phe Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr
Ala305 310 315 320Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr 325 330 335Cys Ala Arg Arg Gly Ile Ala Ala Val Val
Gly Gly Phe Asp Tyr Trp 340 345 350Gly Gln Gly Thr Thr Leu Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro 355 360 365Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 370 375 380Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr385 390 395 400Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 405 410
415Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
420 425 430Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 435 440 445His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Pro Lys Ser 450 455 460Cys Asp Lys46541467PRTHomo sapiens 41Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Phe
20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr Gln
Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly
Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu
Pro Lys Ser Cys 210 215 220Asp Lys Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly225 230 235 240Ser Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly 245 250 255Ala Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp 260 265 270Phe Trp Met
Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp 275 280 285Ile
Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr Gln Lys 290 295
300Phe Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr
Ala305 310 315 320Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr 325 330 335Cys Ala Arg Arg Gly Ile Ala Ala Val Val
Gly Gly Phe Asp Tyr Trp 340 345 350Gly Gln Gly Thr Thr Leu Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro 355 360 365Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 370 375 380Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr385 390 395 400Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 405 410
415Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
420 425 430Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn 435 440 445His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Pro Lys Ser 450 455 460Cys Asp Lys46542704PRTHomo sapiens 42Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Phe
20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr Gln
Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly
Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170
175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Pro Lys Ser Cys 210 215 220Asp Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ile Glu Gly Arg Gly225 230 235 240Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val 245 250 255Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val 260 265
270Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Phe Trp Met
275 280 285Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
Gly Ala 290 295 300Ile Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr Gln
Lys Phe Arg Gly305 310 315 320Arg Ala Thr Leu Thr Ala Asp Lys Ser
Thr Ser Thr Ala Tyr Met Glu 325 330 335Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg 340 345 350Arg Gly Ile Ala Ala
Val Val Gly Gly Phe Asp Tyr Trp Gly Gln Gly 355 360 365Thr Thr Leu
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 370 375 380Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu385 390
395 400Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp 405 410 415Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 420 425 430Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 435 440 445Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 450 455 460Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Pro Lys Ser Cys Asp Lys465 470 475 480Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 485 490 495Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 500 505
510Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
515 520 525Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn 530 535 540Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg Val545 550 555 560Val Ser Val Leu Thr Val Leu Ala Gln
Asp Trp Leu Asn Gly Lys Glu 565 570 575Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys 580 585 590Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 595 600 605Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 610 615 620Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu625 630
635 640Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu 645 650 655Asp Ser Asp Gly Ser Phe Leu Leu Tyr Ser Lys Leu Thr
Val Asp Lys 660 665 670Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met His Glu 675 680 685Ala Leu His Asn Gln Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Pro Gly 690 695 70043329PRTHomo sapiens 43Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Lys Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
32544329PRTHomo sapiens 44Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120
125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235
240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His
Gly Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser
Leu Ser Pro Gly 32545450PRTHomo sapiens 45Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Phe 20 25 30Trp Met Gln Trp
Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile
Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr Gln Lys Phe 50 55 60Arg Gly
Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly 45046450PRTHomo sapiens 46Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30Trp Met Gln Trp Ile
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr
Pro Gly Asp Asp Asp Ala Arg Tyr Thr Gln Lys Phe 50 55 60Arg Gly Arg
Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly
100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215
220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro
Gly 45047450PRTHomo sapiens 47Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30Trp Met Gln Trp Ile Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly
Asp Asp Asp Ala Arg Val Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr
Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410
415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser 435 440 445Pro Gly 45048450PRTHomo sapiens 48Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Trp
Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45Gly Ser Ile Tyr Pro Gly Asp Asp Asp Ala Arg Tyr Thr Gln Lys Phe
50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe
Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310
315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Lys
Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425
430His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445Pro Gly 45049450PRTHomo sapiens 49Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Phe 20 25 30Trp Met Gln
Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala
Ile Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr Gln Lys Phe 50 55 60Arg
Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Lys Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly 45050450PRTHomo sapiens 50Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30Trp Met Gln Trp Ile
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr
Pro Gly Asp Asp Asp Ala Arg Tyr Thr Gln Lys Phe 50 55 60Arg Gly Arg
Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly
100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215
220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330
335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Lys Pro Gln Val
340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro
Gly 45051450PRTHomo sapiens 51Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp Phe 20 25 30Trp Met Gln Trp Ile Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly
Asp Asp Asp Ala Arg Val Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr
Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230
235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Lys Pro Gln Val 340 345
350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445Pro Gly
45052497PRTHomo sapiens 52Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Ile Ser Asn His 20 25 30Leu His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile
Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln 115 120
125Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys
130 135 140Lys Ala Ser Gly Tyr Thr Phe Ser Asp Phe Trp Met Gln Trp
Ile Arg145 150 155 160Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly
Ala Ile Tyr Pro Gly 165 170 175Asp Asp Asp Ala Arg Val Thr Gln Lys
Phe Arg Gly Arg Ala Thr Leu 180 185 190Thr Ala Asp Lys Ser Thr Ser
Thr Ala Tyr Met Glu Leu Ser Ser Leu 195 200 205Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys Ala Arg Arg Gly Ile Ala 210 215 220Ala Val Val
Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr225 230 235
240Val Ser Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly
245 250 255Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys
Lys Thr 260 265 270Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp
Val Lys Gln Arg 275 280 285Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr
Ile Asn Pro Ser Arg Gly 290 295 300Tyr Thr Asn Tyr Asn Gln Lys Phe
Lys Asp Lys Ala Thr Leu Thr Thr305 310 315 320Asp Lys Ser Ser Ser
Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser 325 330 335Glu Asp Ser
Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr 340 345 350Cys
Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val 355 360
365Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val
370 375 380Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala
Ser Pro385 390 395 400Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser
Ser Ser Val Ser Tyr 405 410 415Met Asn Trp Tyr Gln Gln Lys Ser Gly
Thr Ser Pro Lys Arg Trp Ile 420 425 430Tyr Asp Thr Ser Lys Val Ala
Ser Gly Val Pro Tyr Arg Phe Ser Gly 435 440 445Ser Gly Ser Gly Thr
Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala 450 455 460Glu Asp Ala
Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu465 470 475
480Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His His His
485 490 495His53450PRTMus musculus 53Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Ser Asp Phe 20 25 30Trp Met Gln Trp Ile
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr
Pro Gly Asp Asp Asp Ala Arg Val Thr Gln Lys Phe 50 55 60Arg Gly Arg
Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly
100
105 110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro
Ser 115 120 125Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly
Ser Ser Val 130 135 140Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro
Glu Pro Val Thr Leu145 150 155 160Thr Trp Asn Ser Gly Ser Leu Ser
Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Asp Leu
Tyr Thr Leu Ser Ser Ser Val Thr Val Thr 180 185 190Ser Ser Thr Trp
Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro 195 200 205Ala Ser
Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr 210 215
220Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu
Gly225 230 235 240Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys
Asp Val Leu Met 245 250 255Ile Ser Leu Ser Pro Ile Val Thr Cys Val
Val Val Asp Val Ser Glu 260 265 270Asp Asp Pro Asp Val Gln Ile Ser
Trp Phe Val Asn Asn Val Glu Val 275 280 285His Thr Ala Gln Thr Gln
Thr His Arg Glu Asp Tyr Asn Ser Thr Leu 290 295 300Arg Val Val Ser
Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly305 310 315 320Lys
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile 325 330
335Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val
340 345 350Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln
Val Thr 355 360 365Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp
Ile Tyr Val Glu 370 375 380Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn
Tyr Lys Asn Thr Glu Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser
Tyr Phe Met Tyr Ser Lys Leu Arg Val 405 410 415Glu Lys Lys Asn Trp
Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val 420 425 430His Glu Gly
Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr 435 440 445Pro
Gly 45054213PRTMus musculus 54Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Ile Ser Asn His 20 25 30Leu His Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser
Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe
Ala Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Ala Asp Ala Ala Pro 100 105
110Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly
115 120 125Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp
Ile Asn 130 135 140Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn
Gly Val Leu Asn145 150 155 160Ser Trp Thr Asp Gln Asp Ser Lys Asp
Ser Thr Tyr Ser Met Ser Ser 165 170 175Thr Leu Thr Leu Thr Lys Asp
Glu Tyr Glu Arg His Asn Ser Tyr Thr 180 185 190Cys Glu Ala Thr His
Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205Asn Arg Asn
Glu Cys 21055450PRTMus musculus 55Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Ser Asp Tyr 20 25 30Trp Met Gln Trp Ile Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ala Ile Tyr Pro
Gly Asp Asp Asp Ala Arg Tyr Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala
Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser
115 120 125Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser
Ser Val 130 135 140Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu
Pro Val Thr Leu145 150 155 160Thr Trp Asn Ser Gly Ser Leu Ser Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Asp Leu Tyr
Thr Leu Ser Ser Ser Val Thr Val Thr 180 185 190Ser Ser Thr Trp Pro
Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro 195 200 205Ala Ser Ser
Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr 210 215 220Ile
Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly225 230
235 240Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu
Met 245 250 255Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp
Val Ser Glu 260 265 270Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val
Asn Asn Val Glu Val 275 280 285His Thr Ala Gln Thr Gln Thr His Arg
Glu Asp Tyr Asn Ser Thr Leu 290 295 300Arg Val Val Ser Ala Leu Pro
Ile Gln His Gln Asp Trp Met Ser Gly305 310 315 320Lys Glu Phe Lys
Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile 325 330 335Glu Arg
Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val 340 345
350Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr
355 360 365Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr
Val Glu 370 375 380Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys
Asn Thr Glu Pro385 390 395 400Val Leu Asp Ser Asp Gly Ser Tyr Phe
Met Tyr Ser Lys Leu Arg Val 405 410 415Glu Lys Lys Asn Trp Val Glu
Arg Asn Ser Tyr Ser Cys Ser Val Val 420 425 430His Glu Gly Leu His
Asn His His Thr Thr Lys Ser Phe Ser Arg Thr 435 440 445Pro Gly
45056213PRTMus musculus 56Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Ile Ser Asn His 20 25 30Leu His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile
Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Ala Asp Ala Ala Pro 100 105 110Thr
Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 115 120
125Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn
130 135 140Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val
Leu Asn145 150 155 160Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Met Ser Ser 165 170 175Thr Leu Thr Leu Thr Lys Asp Glu Tyr
Glu Arg His Asn Ser Tyr Thr 180 185 190Cys Glu Ala Thr His Lys Thr
Ser Thr Ser Pro Ile Val Lys Ser Phe 195 200 205Asn Arg Asn Glu Cys
21057214PRTHomo sapiens 57Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Ile Ser Asn His 20 25 30Leu His Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Lys Tyr Ala Ser Gln Ser Ile
Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Leu 85 90 95Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu
Cys 21058447PRTHomo sapiens 58Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Trp Met Gln Trp Ile Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ser Ile Tyr Pro Gly
Asp Asp Asp Ala Arg Tyr Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr
Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg
Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105
110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser
Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu
Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205Lys Pro Ser
Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215 220Pro
Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser225 230
235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln
Glu Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val
Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345
350Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Glu Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 44559450PRTHomo
sapiens 59Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Asp Tyr 20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45Gly Ser Ile Tyr Pro Gly Asp Asp Asp Ala Arg
Tyr Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala
Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150
155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Pro Lys Ser Cys 210 215 220Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly225 230 235 240Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265
270Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gln Ser
Thr Tyr 290 295 300Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly305 310 315 320Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 325 330 335Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro385 390
395 400Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
Val 405 410 415Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 420 425 430His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser 435 440 445Pro Gly 45060447PRTHomo sapiens
60Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp
Phe 20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr
Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val
Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215 220Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu Gly 435 440 44561443PRTHomo sapiens
61Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp
Tyr 20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Arg Tyr Thr
Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val
Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val
Ser Ser Gly Pro Ser Val Phe Pro Leu 115 120 125Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys 130 135 140Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser145 150 155
160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser 180 185 190Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His
Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys Arg Val Glu Ser Lys
Tyr Gly Pro Pro Cys Pro 210 215 220Pro Cys Pro Ala Pro Glu Phe Leu
Gly Gly Pro Ser Val Phe Leu Phe225 230 235 240Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255Thr Cys Val
Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280
285Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
290 295 300Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val305 310 315 320Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
Thr Ile Ser Lys Ala 325 330 335Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Gln 340 345 350Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser385 390 395
400Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
405 410 415Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His 420 425 430Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435
44062447PRTHomo sapiens 62Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Trp Met Gln Trp Ile Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ser Ile Tyr Pro Gly Asp
Asp Asp Ala Arg Tyr Thr Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg
Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln
Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120
125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala
130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr Cys Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 210 215 220Pro Pro Cys
Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser225 230 235
240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu Ala Gln
Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330 335Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360
365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Glu Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn Gln Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Leu Gly 435 440 44563447PRTHomo sapiens
63Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp
Phe 20 25 30Trp Met Gln Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45Gly Ala Ile Tyr Pro Gly Asp Asp Asp Ala Arg Val Thr
Gln Lys Phe 50 55 60Arg Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr
Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val
Gly Gly Phe Asp Tyr Trp Gly 100 105 110Gln Gly Thr Thr Leu Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150 155
160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Ser Lys Tyr Gly 210 215 220Pro Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265 270Glu
Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280
285Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
290 295 300Ser Val Leu Thr Val Leu Ala Gln Asp Trp Leu Asn Gly Lys
Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
Ser Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395
400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
405 410 415Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala 420 425 430Leu His Asn Gln Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly 435 440 44564450PRTHomo sapiens 64Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30Trp Met Gln
Trp Ile Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Ser
Ile Tyr Pro Gly Asp Asp Asp Ala Arg Tyr Thr Gln Lys Phe 50 55 60Arg
Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Arg Gly Ile Ala Ala Val Val Gly Gly Phe Asp Tyr Trp
Gly 100 105 110Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
210 215 220Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315
320Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val 340 345 350Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser 355 360 365Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu 370 375 380Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro385 390 395 400Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440
445Pro Gly 450
* * * * *
References